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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. Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays applicable for low platelet counts.

    PubMed

    van Bladel, Esther R; Laarhoven, Annemieke G; van der Heijden, Laila B; Heitink-Pollé, Katja M; Porcelijn, Leendert; van der Schoot, C Ellen; de Haas, Masja; Roest, Mark; Vidarsson, Gestur; de Groot, Philip G; Bruin, Marrie C A

    2014-03-06

    Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 μL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts (<10(9)). These tests show that platelet function is related to bleeding phenotype in chronic ITP.

  3. Development of a high-throughput ELISA assay for platelet function testing using platelet-rich plasma or whole blood.

    PubMed

    Salles, Isabelle; Broos, Katleen; Fontayne, Alexandre; Szántó, Tímea; Ruan, Changgeng; Nurden, Alan T; Vanhoorelbeke, Karen; Deckmyn, Hans

    2010-08-01

    Platelets play an essential role in the development of cardiovascular diseases and are the target of several agents that can inhibit their function. Despite the existence of a wide array of techniques to study platelet function, an assay to evaluate several platelet signalling pathways in a high-throughput fashion, combined with minimal blood volume and handling is still needed. We have developed a sensitive assay in the form of a sandwich ELISA where monoclonal antibodies against P-selectin or alphaIIbbeta3 and GPIbalpha were used to capture and detect platelets, respectively, in the presence of five different agonists [ADP, TRAP (thrombin receptor agonist), U46619 (thromboxane A2 analogue), collagen-related-peptide, and arachidonic acid]. Binding of platelets to the antibodies increased dose-dependently with the concentration of either agonist, while binding of ADP-activated platelets was abrogated when inhibitors of platelet activation were concomitantly added. The test showed good sample reproducibility in 15 healthy donors with conserved platelet response to agonists throughout the assay. Healthy subjects could be identified as normal-, hypo- or hyper-responders for each agonist, which for most cases (73%) was confirmed upon retesting. Finally, we demonstrated that the platelet ELISA assay can not only be used in platelet-rich plasma but also in whole blood; it now awaits large scale studies to assess its full screening and diagnostic values.

  4. The influence of platelets, plasma and red blood cells on functional haemostatic assays.

    PubMed

    Bochsen, Louise; Johansson, Pär I; Kristensen, Annemarie T; Daugaard, Gedske; Ostrowski, Sisse R

    2011-04-01

    Functional whole blood haemostatic assays are used increasingly to guide transfusion therapy and monitor medical treatment and are also applied for in-vitro evaluations of the haemostatic potential of stored platelets. We investigated how the cellular and plasmatic elements, both isolated and combined, influenced the two methodologically different assays, thrombelastography (TEG) and impedance aggregometry (Multiplate). Platelet-rich plasma (200 × 10/l) or pure plasma (0 platelets), with and without added red blood cells (RBCs), hematocrit 0, 0.15 or 0.29, were produced in vitro from platelet concentrates, fresh frozen plasma and stored RBC. Pure platelets were investigated by removing plasma components from platelet concentrates by diafiltration against the platelet storage solution Intersol. Plasma was readded by diafiltration against plasma in Intersol. Haemostatic function was evaluated by TEG and Multiplate. In the TEG, increasing amounts of RBC reduced clot strength and clot kinetics (α-angle), most markedly in plasma/RBC without platelets. In contrast, RBC in a platelet concentrate matrix enhanced Multiplate aggregation in response to weak agonists (ADP and arachidonic acid). Furthermore, removing plasma from platelet concentrates eliminated the TEG response and diminished the Multiplate aggregation response, but readding plasma to the pure platelet concentrates restored the response. Each of the elements in whole blood, plasma, platelets and RBC, affected the Multiplate and TEG results differently. The results emphasize that the concentrations of all cellular and plasmatic components in whole blood should be taken into account when interpreting results obtained by TEG and multiplate.

  5. Functional assay of antiplatelet drugs based on margination of platelets in flowing blood

    PubMed Central

    Eichinger, Colin D.; Fogelson, Aaron L.; Hlady, Vladimir

    2016-01-01

    A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could “prime” the marginated platelet subpopulation for enhanced activation and adhesion downstream. By creating an upstream surface patch with an immobilized platelet agonist, platelet flow margination was used to test how effective antiplatelet drugs are in suppressing downstream platelet activation and adhesion. The platelet adhesion downstream was measured by a so-called “capture” patch region close to the distal end of the flow chamber. Platelet adhesion downstream was found to be dose-dependent on the upstream surface coverage of the “priming” patch, with immobilized fibrinogen acting as a platelet agonist. Several antiplatelet agents (acetylsalicylic acid, eptifibatide, and tirofiban) were evaluated for their efficacy in attenuating downstream adhesion after upstream platelet priming. The activation of the platelet population was found to be dependent on both the extent of the upstream agonist stimulus and the antiplatelet drug concentration. Such a relationship provides an opportunity to measure the efficacy of specific antiplatelet agents against the type and concentration of upstream platelet agonists. PMID:27030476

  6. 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.

  7. Platelet Function Tests.

    PubMed

    Lordkipanidzé, Marie

    2016-04-01

    Traditionally developed for diagnosis of bleeding disorders, platelet function assays have become increasingly used in basic research on platelet physiology, in phenotype-genotype associations in bleeding disorders, in drug development as surrogate endpoints of efficacy of new antiplatelet therapy, and to an extent, in the monitoring of antiplatelet therapy in clinical practice to predict thrombotic and bleeding risk. A multiplicity of platelet function assays is available to measure the level of platelet activity in various settings. These include assays that are restricted to a specialized laboratory as well as point-of-care instruments meant to investigate platelet function at patient bedside. Unlike tests that determine a defined quantity or measurement of a clinical biomarker (e.g., cholesterol or blood pressure), platelet function testing assesses the dynamics of living cells, which immediately presents a series of unique problems to any laboratory or clinic. This article presents currently used platelet function assays and discusses important variables to take into account when performing these assays, including preanalytical issues and difficulties in interpreting platelet function test results.

  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. Functional fractionation of platelets.

    PubMed

    Haver, V M; Gear, A R

    1981-02-01

    Studies of platelet populations suggest that they are heterogeneous in size, age, and metabolic parameters. In an attempt to correlate these parameters with efficiency of aggregation, a new technique, functional fractionation, was developed. Platelet populations are separated by their differential reactivity to aggregating agents. For example, low doses of ADP (0.1 to 0.7 microM) are added to stirred PRP, after which gentle centrifugation is used to remove aggregates from single unreacted platelets. The loose aggregates can be readily dispersed for comparison of the physical or biochemical properties of the reacted versus unreacted platelets. It was found that reactive platelets were larger (6.5 micrometer3) than unreacted platelets (5.51 micrometer3). No significant difference in density existed between the two populations, and no release of [14C]serotonin from prelabeled platelets occurred during functional fractionation. Scanning and transmission electron microscopy confirmed the size difference and revealed that in both populations platelets were structurally intact with a normal discoid shape and no significant difference in organelle content. Human platelets most reactive to ADP were also enriched in glycogen (3.6-fold), ATP (1.6-fold), and ADP (twofold), compared with less reactive cells. These "reactive" cells took up more 51[Cr] and contained 1.9 times more surface sialic acid. In an in vivo aging experiment, rats were injected with 75[Se]methionine. Shortly after labeling (1 day), the most reactive platelets possessed the highest amount of 75[Se]. These results reveal that functionally active platelets, which are also larger, are more active metabolically than less reactive platelets, possess a higher negative surface charge, and may be a younger population.

  10. Platelet function tests: a comparative review.

    PubMed

    Paniccia, Rita; Priora, Raffaella; Liotta, Agatina Alessandrello; 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.

  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. 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

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

    PubMed

    Rodgers, G M

    1999-06-01

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

  14. Detection of platelet alloimmunity with a platelet-associated IgG assay

    SciTech Connect

    Myers, T.J.; Kim, B.K.; Steiner, M.; Bishop, J.; Baldini, M.G.

    1981-06-01

    A quantitative immunofluorescence PA-IgG assay was used to detect alloimmunity to platelets. The assay identified serum alloantibodies in 10 out of 14 multitransfused patients and for two of three infants with neonatal thrombocytopenia. The correct separation of all multitransfused patients into alloimmune and nonalloimmune groups by the PA-IgG assay was substantiated with chromium-51-labeled platelet survival studies. The allogeneic nature of the serum antibodies was demonstrated by progressive absorption of the antibody with increasing numbers of allogeneic platelets but not with autologous platelets. The sensitivity of the PA-IgG assay for detection of serum alloantibodies was superior to that of platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing. In dilution experiments with alloimmune serum, elevated levels of serum PA-IgG could still be detected on donor platelets when platelet aggregation and serotonin release tests became negative. Platelet survival studies with selected platelets performed in the 10 alloimmunized, multitransfused patients confirmed the results of the PA-IgG assays, predicting alloimmunity to the donor platelets. In contrast, platelet aggregation, platelet serotonin release, and lymphocytotoxicity testing indicated alloimmunity for 50% or less of the patients. Reduced platelet survival times were also seen with HLA A- and HLA B-matched donor platelets when donor-recipient incompatibility was demonstrated by the PA-IgG assay. Thus the PA-IgG assay provides a sensitive method to detect serum platelet alloantibodies and may offer a technique in platelet crossmatching.

  15. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2013-10-01

    mice and mice transfused with Syk inhibitor-treated platelets . Platelet lodging was remarkably decreased in lungs of mice transfused with Syk...AD_________________ Award Number: W81XWH-12-1-0523 TITLE: Complement Activation Alters Platelet ...30September2012–29September2013 4. TITLE AND SUBTITLE Complement Activation Alters Platelet Function 5a. CONTRACT NUMBER W81XWH-12-1-0523 5b. GRANT NUMBER

  16. New trends in bioanalytical microdevices to assess platelet function.

    PubMed

    Basabe-Desmonts, Lourdes; Meade, Gerardene; Kenny, Dermot

    2010-10-01

    Cardiovascular disease is the major cause of mortality globally. The role of platelets and antiplatelet drugs in the treatment of cardiovascular disease is widely appreciated. Platelets have a less well-known role in cancer and inflammation and as the role of platelets in cancer and inflammation is increasingly understood, there is a compelling need to develop diagnostic assays of platelet function to guide clinical management. Most current platelet-function tests are focused on platelet aggregation, are cumbersome, require dedicated laboratory personnel, measure a single response to a single agonist and artificially separate platelets from blood, thus, these assays do not reflect the pathophysiolgical environment of complex disease states. New technology platforms are emerging that address the physiological adhesive function of platelets to vascular-specific matrices using small volumes of whole blood, giving rapid results. These technologies will guide therapy in the prevention of cardiovascular disease and probably in risk management in both cancer and atherosclerosis.

  17. Selection of donor platelets for alloimmunized patients using a platelet-associated IgG assay

    SciTech Connect

    Myers, T.J.; Kim, B.K.; Steiner, M.; Baldini, M.G.

    1981-09-01

    A quantitative immunofluorescence platelet-associated immunoglobulin-G (PA-IgG) assay was used to detect alloimmunity to platelets in 8/12 multitransfused patients and to perform platelet crossmatching in the 8 alloimmunized patients. The correct separation of multitransfused patients into alloimmune and nonalloimmune groups was substantiated with chromium-51-labeled platelet survival studies. For 5 alloimmunized patients, compatible and incompatible donor platelets were demonstrated by PA-IgG crossmatching and were confirmed by platelet survival studies. With the other 3 alloimmunized patients, only Pa-IgG incompatible donor platelets were found. Survival studies with 5 of these incompatible donor platelets showed markedly reduced survival times on 4 occasions. Pa-IgG compatible donor platelets survived 3.5 to 8.7 days, while Pa-IgG incompatible platelets showed survival times of 0.1 to 2.4 days.

  18. 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.

  19. [The usefulness of platelet function evaluation in clinical practice].

    PubMed

    Elalamy, Ismail; Gkalea, Vasiliki; Gerotziafas, Grigorios; Ketatni, Hela; Hatmi, Mohamed

    2013-11-01

    Platelets play a pivotal role in the regulation of both thrombosis and haemostasis. Functional testing of platelet response has been exclusively used in the diagnosis and management of bleeding disorders. Recent advances of light transmission aggregometry and development of more useful devices have demonstrated the clinical utility to enlarge platelet function testing in patients with cardiovascular disease. The ex vivo measurement of residual platelet response seems, with some assays, predictive of adverse clinical events. Still a debate, it represents an emerging area of interest for both the clinician and the basic scientist. Heparin-induced thrombocytopenia diagnosis is also difficult and a functional assay is now available for an easier and rapid method to rule out such a life-threatening situation. This review article will describe the available methods of measuring platelet response and will discuss both the limitations and emerging data supporting the role of platelet function studies in clinical practice.

  20. Platelet serotonin modulates immune functions.

    PubMed

    Mauler, M; Bode, C; Duerschmied, D

    2016-01-01

    This short review addresses immune functions of platelet serotonin. Platelets transport serotonin at a high concentration in dense granules and release it upon activation. Besides haemostatic, vasotonic and developmental modulation, serotonin also influences a variety of immune functions (mediated by different serotonin receptors). First, platelet serotonergic effects are directed against invading pathogens via activation and proliferation of lymphocytes, modulation of cytokine release, and recruitment of neutrophils to sites of acute inflammation by induction of selectin expression on endothelial cells. Second, serotonin levels are elevated in autoimmune diseases, such as asthma or rheumatoid arthritis, and during tissue regeneration after ischemia of myocardium or brain. Specific antagonism of serotonin receptors appears to improve survival after myocardial infarction or sepsis and to attenuate asthmatic attacks in animal models. It will be of great clinical relevance if these findings can be translated into human applications. In conclusion, targeting immune modulatory effects of platelet serotonin may provide novel therapeutic options for common health problems.

  1. Point-of-care platelet function assays demonstrate reduced responsiveness to clopidogrel, but not aspirin, in patients with Drug-Eluting Stent Thrombosis whilst on dual antiplatelet therapy

    PubMed Central

    Hobson, Alex R; Petley, Graham; Morton, Geraint; Dawkins, Keith D; Curzen, Nick P

    2008-01-01

    Background To test the hypothesis that point-of-care assays of platelet reactivity would demonstrate reduced response to antiplatelet therapy in patients who experienced Drug Eluting Stent (DES) ST whilst on dual antiplatelet therapy compared to matched DES controls. Whilst the aetiology of stent thrombosis (ST) is multifactorial there is increasing evidence from laboratory-based assays that hyporesponsiveness to antiplatelet therapy is a factor in some cases. Methods From 3004 PCI patients, seven survivors of DES ST whilst on dual antiplatelet therapy were identified and each matched with two patients without ST. Analysis was performed using (a) short Thrombelastogram PlateletMapping™ (TEG) and (b) VerifyNow Aspirin and P2Y12 assays. TEG analysis was performed using the Area Under the Curve at 15 minutes (AUC15) as previously described. Results There were no differences in responses to aspirin. There was significantly greater platelet reactivity on clopidogrel in the ST group using the Accumetrics P2Y12 assay (183 ± 51 vs. 108 ± 31, p = 0.02) and a trend towards greater reactivity using TEG AUC15 (910 ± 328 vs. 618 ± 129, p = 0.07). 57% of the ST group by TEG and 43% of the ST cases by Accumetrics PRU had results > two standard deviations above the expected mean in the control group. Conclusion This study demonstrates reduced platelet response to clopidogrel in some patients with DES ST compared to matched controls. The availability of point-of-care assays that can detect these responses raises the possibility of prospectively identifying DES patients at risk of ST and manipulating their subsequent risk. PMID:18312665

  2. Reelin is a platelet protein and functions as a positive regulator of platelet spreading on fibrinogen.

    PubMed

    Tseng, Wei-Lien; Huang, Chien-Ling; Chong, Kowit-Yu; Liao, Chang-Huei; Stern, Arnold; Cheng, Ju-Chien; Tseng, Ching-Ping

    2010-02-01

    Abnormalities of platelet functions have been linked to reelin-impaired neuronal disorders. However, little attention has been given to understanding the interplay between reelin and platelet. In this study, reelin was found to present in the human platelets and megakaryocyte-like leukemic cells. Reelin-binding assays revealed that extracellular reelin can interact with platelets through the receptor belonging to the low density lipoprotein receptor gene family. The reelin-to-platelet interactions enhance platelet spreading on fibrinogen concomitant with the augmentation of lamellipodia formation and F-actin bundling. In contrast, reelin has no effect on integrin alphaIIbbeta3 activation and agonist-induced platelet aggregation. Molecular analysis revealed that the up-regulation of Rac1 activity and the inhibition of protein kinase C delta-Thr505 phosphorylation are important for reelin-mediated enhancement of platelet spreading on fibrinogen. These findings demonstrate for the first time that reelin is present in platelets and the reelin-to-platelet interactions play a novel role in platelet signaling and functions.

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

    PubMed

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

    2013-11-07

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

  4. The Platelet Function Defect of Cardiopulmonary Bypass.

    DTIC Science & Technology

    1992-11-24

    fibrinolytic and coagulation systems occur during CPB,1 a platelet function defect is generally considered to be the primary CPB-induced hemostatic...platelets.39 OKM5 (provided by Dr. Patricia Rao, Ortho Diagnostic Systems , Raritan, NJ) is directed against platelet membrane GPIV.40 Flow Cytometric...22 after degranulation.7-14-16-18 Utilizing washed platelet systems , Nieuwenhuis et al.14 found a modest increase during CPB of the platelet

  5. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders.

    PubMed

    Mumford, Andrew D; Frelinger, Andrew L; Gachet, Christian; Gresele, Paolo; Noris, Patrizia; Harrison, Paul; Mezzano, Diego

    2015-07-01

    Measurement of platelet granule release to detect inherited platelet secretion disorders (IPSDs) is essential for the evaluation of patients with abnormal bleeding and is necessary to distinguish which granule sub-types are affected and whether there is abnormal granule bio-synthesis or secretion. The radioactive serotonin incorporation and release assay, described before 1970, is still considered the "gold standard" test to assess platelet δ-granule release, although is unsuitable for clinical diagnostic laboratories. Luciferin-based assays, such as lumiaggregometry, are the most widely performed alternatives, although these methods do not distinguish defects in δ-granule biosynthesis from defects in secretion. Platelet α-granule release is commonly evaluated using flow cytometry by measuring surface exposure of P-selectin after platelet activation. However, this assay has poor sensitivity for some α-granule disorders. Only few studies have been published with more recently developed assays and no critical reviews on these methods are available. In this review, we describe the rationale for developing robust and accurate laboratory tests of platelet granule release and describe the characteristics of the currently available tests. We identify an unmet need for further systematic evaluation of new assays and for standardisation of methodologies for clinical diagnostic laboratories.

  6. 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.

  7. Shear-mediated platelet activation in patients implanted with continuous flow LVADs: A preliminary study utilizing the platelet activity state (PAS) assay.

    PubMed

    Valerio, Lorenzo; Consolo, Filippo; Bluestein, Danny; Tran, Phat; Slepian, Marvin; Redaelli, Alberto; Pappalardo, Federico

    2015-08-01

    Left ventricular assist devices (LVADs) have emerged as vital life-saving therapeutic systems for patients with advanced and end-stage heart failure (HF). Despite their efficacy, VAD systems remain limited by post-implantation thrombotic complications. Shear-mediated platelet activation is the major driver of such complications in these devices. Nowadays few platelet function assays are routinely utilized in assessing the degree of platelet activation in VAD implanted patients. No assays exist that specifically target shear-mediated platelet activation. The platelet activity state (PAS) is a novel assay that has been well validated in vitro, measuring thrombin release as a surrogate for shear-mediated platelet activation. To date limited data exist as to the utility of this assay in the clinical setting. In the present study we evaluated eight LVAD patients' platelet activation level using the PAS assay. Simultaneous measurements of conventional prothrombotic and hemolysis markers, - i.e. fibrinogen and lactate dehydrogenase (LDH) - were also performed. Trends as to alteration from baseline were studied. We observed that the PAS assay allowed detection of an abnormal level of platelet activation in one patient in our series who suffered from an overt thrombosis. Interestingly in the same patient no signal of major abnormality in fibrinogen or LDH was detected. Further for 7/8 patients who were free of thrombosis, no significant level of platelet activation was detected via PAS assay, while elevation in fibrinogen and LDH were observed. As such, from our observational series it appears that the PAS assay is a sensitive and specific indicator of shear-mediated platelet activation. Further patients' experience will help elucidate the role of this promising assay in the management of LVAD implanted patients.

  8. 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

  9. Platelet function and constituents of platelet rich plasma.

    PubMed

    Pelletier, M H; Malhotra, A; Brighton, T; Walsh, W R; Lindeman, R

    2013-01-01

    Platelet Rich Plasma (PRP) therapies require blood to be processed prior to application, however, the full assessment of the output of platelet sequestration devices is lacking. In this study the products of the Autologous Fluid Concentrator (Circle BiologicsTM, Minneapolis, MN) and the Gravitational Platelet Separation System (GPS, Biomet, Warsaw, IN, USA) were evaluated in terms of platelet viability and PRP constituents. The AFC and GPS produced 6.4 (±1.0) ml and 6.3 (±0.4) ml of PRP, with platelet recovery of 46.4% (±14.7%) and 59.8% (±24.2%) producing fold increases of platelets of 4.19 (±1.62) and 5.19 (±1.62), respectively. Fibrinogen concentration was increased above baseline PPP produced with the AFC. pH was lower for both of the processed samples than for whole blood. White Blood Cell count was increased around 5 fold. Functional tests showed preserved viability with both devices. This represents essential knowledge that every treating physician should have before they can confidently administer PRP therapy produced by any method. These are the first published results of platelet function for the GPS system and the first performance results of the AFC system. The PRP produced is classified according to broad classifications as Leukocyte-PRP (L-PRP) for both devices.

  10. [Cardiology. Platelet function testing for clinicians].

    PubMed

    Pellaton, Cyril; Eeckhout, Eric; Silvain, Johanne; Montalescot, Gilles; Collet, Jean-Phillipe

    2014-01-15

    Platelet P2YI2 receptor inhibition with clopidogrel, prasugrel or ticagrelor plays a key role to prevent recurrent ischaemic events after percutaneous coronary intervention in acute coronary syndromes or elective settings. The degree of platelet inhibition depends on the antiplatelet medication used and is influenced by clinical and genetic factors. A concept of therapeutic window exists. On one side, efficient anti-aggregation is required in order to reduce cardio-vascular events. On the other side, an excessive platelet inhibition represents a risk of bleeding complications. This article describes the current knowledge about some platelet function tests and genetic tests and summarises their role in the clinical practice.

  11. 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

  12. Serum-induced platelet procoagulant activity: an assay for the characterization of prothrombotic disorders.

    PubMed

    Warner, M N; Pavord, S; Moore, J C; Warkentin, T E; Hayward, C P; Kelton, J G

    1999-02-01

    Platelets contribute to hemostasis by forming a platelet plug and by providing a procoagulant surface for the assembly and activation of the coagulation factors. The contribution of platelets to prothrombotic disorders has been difficult to analyze. Recently an assay was reported that measured the procoagulant activity of test platelets by making the platelet lipid surface the limiting factor in the production of thrombin. In this report we describe a novel technique, based on this assay, that we used to study patient serum factors that activate control platelets and in turn initiate measurable procoagulant activity. Using this assay we investigated a group of patients with prothrombotic disorders. The patient test serum was incubated with normal platelets in the presence of activated factor Xa. The resultant thrombin was measured in a chromogenic assay. The rate-limiting step was the presence of any potential platelet-activating factors, such as antibodies in the heat-treated test serum, that would allow the Xa to bind to the platelet phospholipid surface. Serum samples from patients with heparin-induced thrombocytopenia (HIT) and the anti-phospholipid antibody syndrome enhanced platelet procoagulant activity, while samples from patients with idiopathic thrombocytopenic purpura and disseminated intravascular coagulation (DIC) did not. HIT serum samples also induced platelet activation, as measured by platelet microparticle shedding, carbon 14-labeled serotonin release, and platelet aggregation. The measurement of serum-induced platelet procoagulant activity provides a method for the investigation of circulating platelet agonists in prothrombotic disorders.

  13. Modulatory effect of coffee on platelet function.

    PubMed

    Bhaskar, Shobha; Rauf, Arun A

    2010-01-01

    Blood platelets play a major role in cardiovascular disease (CVD) and thrombosis. Conflicting information exists regarding the effect of coffee consumption on the cardiovascular system. We have investigated whether the consumption of moderate amount of coffee affect platelet functions and primary hemostasis in vivo in normal and high fat diet fed rats. Coffee fed group showed significant (P < 0.05) decrease in mean platelet volume, platelet crit and platelet distribution width as compared to high fat diet (HFD) group. The concentration of malondialdehyde in platelets increased in atherosclerotic group indicates the increased thromboxane A2 (TXA2) production from membrane arachidonic acid and it was decreased in coffee treated group. Platelet aggregation studies with ADP, collagen, arachidonic acid and epinephrine showed significant (P < 0.05) decrease in aggregation in coffee fed group. Scanning electron microscopic studies revealed that platelet aggregation tendency increased in HFD group and was reduced in coffee fed group. These results indicate that coffee is active in inhibiting platelet aggregation, a critical step involved in thrombosis.

  14. Platelet function in Takotsubo cardiomyopathy.

    PubMed

    Núñez-Gil, Iván J; Bernardo, Esther; Feltes, Gisela; Escaned, Javier; Mejía-Rentería, Hernán D; De Agustín, José Alberto; Vivas, David; Nombela-Franco, Luis; Jiménez-Quevedo, Pilar; Macaya, Carlos; Fernández-Ortiz, Antonio

    2015-05-01

    Takotsubo cardiomyopathy (TK) includes a transient left ventricular dysfunction without obstructive coronary disease, sometimes after stressful situations with elevated cathecolamines. Since catecholamines activate platelets we aimed to study the platelet influence in a TK setting. We included 32 patients with a TK diagnosis, 13 with an acute coronary syndrome (ACS) and 18 healthy volunteers. Once consent informed was obtained, blood samples were extracted and processed (at admission and after 3 months follow-up). Clinical, ecg, echocardiographic and angiographic features were thoroughly recorded.Previous treatment before admission was similar between groups. No differences were observed in clinical features or any of the acute markers studied regarding platelet reactivity between TK compared to ACS. After follow-up, aggregation levels and platelet reactivity showed differences, mainly due to the antithrombotic therapy prescribed at discharge, but similar to volunteers. Circulating epinephrine during the acute phase was significantly higher in TK (p < 0.001). Patients with higher levels of epinephrine had elevated platelet activation and aggregation after 3 months. No differences were observed in Takotsubo acute platelet aggregation compared to patients with ACS, in spite of higher blood levels of adrenaline. Takotsubo patients had elevated platelet aggregation and activation compared with ACS patients at 3 months follow-up because they were less frequently on chronic clopidogrel and ASA. However, they had similar platelet aggregation and activation levels to healthy volunteers despite treatment with low-dose ASA. Takotsubo patients who had higher levels of adrenaline in the acute phase displayed increased platelet reactivity during follow-up.

  15. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2014-10-01

    Negative T cells than B6.lpr mice. This suggests that the absence of PF4 alleviates some tissue damage in the lupus prone mice. 6...mice with PF4-/- mice may alleviate multi organ dysfunction in Lupus prone mice. Reportable Outcomes Nothing to report Conclusions We have...dysfunction in lupus models. We have evaluated the relationship between Syk and platelets and have thus far identified a role for Syk in platelet lodging in

  16. Daily prickly pear consumption improves platelet function.

    PubMed

    Wolfram, R; Budinsky, A; Efthimiou, Y; Stomatopoulos, J; Oguogho, A; Sinzinger, H

    2003-07-01

    Prickly pear is traditionally used by Pima Indians as a dietary nutrient against diabetes mellitus. We examined the effect of daily consumption of 250 g in 8 healthy volunteers and 8 patients with mild familial heterozygous hypercholesterolemia on various parameters of platelet function. Beside its action on lipids and lipoproteins, prickly pear consumption significantly reduced the platelet proteins (platelet factor 4 and beta-thromboglobulin), ADP-induced platelet aggregation and improved platelet sensitivity (against PGI2 and PGE1) in volunteers as well as in patients. Also plasma 11-DH-TXB2 and the WU-test showed a significant improvement in both patients and volunteers. In contrast, collagen-induced platelet aggregation and the number of circulating endothelial cells showed a significant response in patients only. No influence of prickly pear ingestion on peripheral platelet count was monitored. The dietary run-in period did not influence any of the parameters of haemostasis examined. No sex difference was seen. Prickly pear may induce at least part of its beneficial actions on the cardiovascular system via decreasing platelet activity and thereby improving haemostatic balance.

  17. Inherited Platelet Function Disorders: Algorithms for Phenotypic and Genetic Investigation.

    PubMed

    Gresele, Paolo; Bury, Loredana; Falcinelli, Emanuela

    2016-04-01

    Inherited platelet function disorders (IPFDs) manifest with mucocutaneous bleeding and are frequently difficult to diagnose due to their heterogeneity, the complexity of the platelet activation pathways and a lack of standardization of the platelet function laboratory assays and of their use for this purpose. A rational diagnostic approach to IPFDs should follow an algorithm where clinical examination and a stepwise laboratory evaluation play a crucial role. A streamlined panel of laboratory tests, with consecutive steps of increasing level of complexity, allows the phenotypic characterization of most IPFDs. A first-line diagnosis of a significant fraction of the IPFD may be made also at nonspecialized centers by using relatively simple tests, including platelet count, peripheral blood smear, light transmission aggregometry, measurement of platelet granule content and release, and the expression of glycoproteins by flow cytometry. Some of the most complex, second- and third-step tests may be performed only in highly specialized laboratories. Genotyping, including the widespread application of next-generation sequencing, has enabled discovery in the last few years of several novel genes associated with platelet disorders and this method may eventually become a first-line diagnostic approach; however, a preliminary clinical and laboratory phenotypic characterization nowadays still remains crucial for diagnosis of IPFDs.

  18. 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.

  19. Effects of drugs on platelet function.

    PubMed

    Morse, E E

    1977-01-01

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

  20. Proteomic approaches to dissect platelet function: half the story

    PubMed Central

    Gnatenko, Dmitri V.; Perrotta, Peter L.; Bahou, Wadie F.

    2006-01-01

    Platelets play critical roles in diverse hemostatic and pathologic disorders and are broadly implicated in various biological processes that include inflammation, wound healing, and thrombosis. Recent progress in high-throughput mRNA and protein profiling techniques has advanced our understanding of the biological functions of platelets. Platelet proteomics has been adopted to decode the complex processes that underlie platelet function by identifying novel platelet-expressed proteins, dissecting mechanisms of signal or metabolic pathways, and analyzing functional changes of the platelet proteome in normal and pathologic states. The integration of transcriptomics and proteomics, coupled with progress in bioinformatics, provides novel tools for dissecting platelet biology. In this review, we focus on current advances in platelet proteomic studies, with emphasis on the importance of parallel transcriptomic studies to optimally dissect platelet function. Applications of these global profiling approaches to investigate platelet genetic diseases and platelet-related disorders are also addressed. PMID:16926286

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

    PubMed

    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

    2016-04-01

    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 α(IIb)β3 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 α(IIb)β3 Markedly, in thrombi from mice deficient in transglutaminase factor XIII, platelet-driven fibrin formation and Rhod-A14 binding were abolished by blockage of integrin α(IIb)β3. Vice versa, star-like fibrin formation from platelets of a patient with deficiency in α(IIb)β3(Glanzmann thrombasthenia) was abolished upon blockage of transglutaminase activity. We conclude that coated platelets, with initial α(IIb)β3 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 α(IIb)β3.

  2. 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

  3. 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.

  4. Validation of a P2Y12-receptor specific whole blood platelet aggregation assay.

    PubMed

    Amann, Michael; Ferenc, Miroslaw; Valina, Christian M; Bömicke, Timo; Stratz, Christian; Leggewie, Stefan; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-11-01

    Testing of P2Y12-receptor antagonist effects can support clinical decision-making. However, most platelet function assays use only ADP as agonist which is not P2Y12-receptor specific. For this reason P2Y12-receptor specific assays have been developed by adding prostaglandin E1 (PGE1) to reduce ADP-induced platelet activation via the P2Y1-receptor. The present study sought to evaluate a P2Y12-receptor specific assay for determination of pharmacodynamic and clinical outcomes. This study enrolled 400 patients undergoing coronary stenting after loading with clopidogrel or prasugrel. ADP-induced platelet reactivity was assessed by whole blood aggregometry at multiple time points with a standard ADP assay (ADPtest) and a P2Y12-receptor specific assay (ADPtest HS, both run on Multiplate Analyzer, Roche Diagnostics). Patients were clinically followed for 1 month and all events adjudicated by an independent committee. In total, 2084 pairs of test results of ADPtest and ADPtest HS were available showing a strong correlation between results of both assays (r = 0.96, p < 0.001). These findings prevailed in multiple prespecified subgroups (e.g., age; body mass index; diabetes). Calculated cutoffs for ADPtest HS and the established cutoffs of ADPtest showed a substantial agreement for prediction of ischemic and hemorrhagic events with a Cohen's κ of 0.66 and 0.66, respectively. The P2Y12-receptor specific ADPtest HS assay appears similarly predictive for pharmacodynamic and clinical outcomes as compared to the established ADPtest assay indicating its applicability for clinical use. Further evaluation in large cohorts is needed to determine if P2Y12-receptor specific testing offers any advantage for prediction of clinical outcome.

  5. Complement Activation Alters Platelet Function

    DTIC Science & Technology

    2015-12-01

    2004; Danese et al., 2003). Recent studies have demonstrated a role for platelets in the development of both innate and adaptive immune responses...mediated modulation of adaptive immunity. A communication link between innate and adaptive immune compartments. Immunity 19:9-19. 4. Fleming, S.D., M...Monestier, and G.C. Tsokos. 2004. Accelerated ischemia/reperfusion- induced injury in autoimmunity-prone mice. Journal of immunology 173:4230-4235

  6. 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.

  7. 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.

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

    PubMed Central

    Barnard, Sunelle A.; Loots, Du Toit; Rijken, Dingeman C.

    2017-01-01

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

  9. Platelets in leucocyte recruitment and function.

    PubMed

    Rossaint, Jan; Zarbock, Alexander

    2015-08-01

    Platelets have a longstanding recognition as an essential cellular component of the coagulation system. However, substantial research over the last decade has added another important aspect to platelet function in that they are also an integral part of the innate immune system. Complex organisms are facing a constant threat of infections by invading pathogens, and they have developed a sophisticated and elegant measure to combat this threat, namely the immune system. Leucocyte recruitment to sites of infections is an essential step at the forefront of the immune response. Platelets have been shown to be involved in several steps of this process and they are an integrated connecting element among haemostasis, host defence, and additional immunological functions (e.g. neutrophil extracellular traps formation). However, the immune system also requires a tight regulation, as an overshooting immune response carries the risk of harming the host itself. This review aims at highlighting the unique features and molecular mechanisms that allow for the interactions of platelets and leucocytes and the regulation of this process. Furthermore, this article identifies the functional relevance of these events for the immune response.

  10. Size Dependent Platelet Subpopulations: Relationship of Platelet Volume to Ultrastructure Enzymatic Activity, and Function.

    DTIC Science & Technology

    1983-03-10

    of the present apheresis instruments to separate the larger more functional platelets from the smaller ones. The selective isolation of large... PLATELET VOLUME T. -(U) BOSTON UNIV MA SCHOOL OF I MEDICINE C B THOMPSON ET RL 10 MAR 83 BUSM-93-89 UNIIDN919CA89 /68 6ilfflfllflflflflll l...N00014-79-C-0168 TECHNICAL REPORT NO. 83-08 SIZE DEPENDENT PLATELET SUBPOPULATIONS: RELATIONSHIP OF PLATELET VOLUME TO ULTRASTRUCTURE. ENZYMATIC ACTIVITY

  11. 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.

  12. Function of eltrombopag-induced platelets compared to platelets from control patients with immune thrombocytopenia.

    PubMed

    Haselboeck, Johanna; Kaider, Alexandra; Pabinger, Ingrid; Panzer, Simon

    2013-04-01

    Data on the in vivo function of platelets induced by the thrombopoietin receptor agonist eltrombopag are scarce. To assess a possible influence of eltrombopag we compared platelet function of eltrombopag-treated immune thrombocytopenia (ITP) patients (group 1; n=10) after treatment response to that from control ITP patients (group 2; n=12). We further analysed platelet function at baseline and after one, three, and four weeks of eltrombopag treatment and estimated daily changes of platelet function during the eltrombopag-induced platelet rise. The formation of platelet-monocyte aggregates (PMA), P-selectin expression [MFI], and platelet adhesion under high shear conditions (surface coverage, SC) in vivo and after in vitro addition of agonists (ADP, TRAP-6, Collagen) were similar between both groups after response to eltrombopag treatment. Only TRAP-6 induced a lower SC in the eltrombopag group (p=0.03). All platelet function parameters except for Collagen-induced P-selectin expression changed significantly during treatment with eltrombopag. PMA, naïve and after addition of ADP or TRAP-6 increased with increasing platelet counts. P-selectin expression decreased, when measured without and upon addition of ADP, increased in the presence of TRAP-6, and remained unchanged after addition of Collagen. SC increased during the eltrombopag-induced platelet rise. All significant changes of platelet function correlated to changes in platelet counts. Two patients developed venous thromboses during eltrombopag treatment, but no association with any distinct single platelet function parameter or combinations thereof was identifiable. Thus, eltrombopag-induced platelets function similar to those from control ITP patients without discernible increased hyper-reactivity.

  13. 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.

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

    PubMed Central

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

    2015-01-01

    Summary 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 five days 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. PMID:26683619

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

    PubMed

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

    2012-01-01

    Fibrinolysis dysfunctions cause bleeding or predisposition to thrombosis. Platelets contain several factors of the fibrinolytic system, which could up or down regulate this process. However, the temporal relationship and relative contributions of plasma and platelet components in clot lysis are mostly unknown. We developed a clot lysis time (CLT) assay in platelet-rich plasma (PRP-CLT, with and without stimulation) and compared it to a similar one in platelet-free plasma (PFP) and to another previously reported test in platelet-poor plasma (PPP). We also studied the differential effects of a single dose of tranexamic acid (TXA) on these tests in healthy subjects. PFP- and PPP-CLT were significantly shorter than PRP-CLT, and the three assays were highly correlated (p < 0.0001). PFP- and PPP-, but more significantly PRP-CLT, were positively correlated with age and plasma PAI-1, von Willebrand factor, fibrinogen, LDL-cholesterol, and triglycerides (p < 0.001). All these CLT assays had no significant correlations with platelet aggregation/secretion, platelet counts, and pro-coagulant tests to explore factor X activation by platelets, PRP clotting time, and thrombin generation in PRP. Among all the studied variables, PFP-CLT was independently associated with plasma PAI-1, LDL-cholesterol, and triglycerides and, additionally, stimulated PRP-CLT was also independently associated with plasma fibrinogen. A single 1 g dose of TXA strikingly prolonged all three CLTs, but in contrast to the results without the drug, the lysis times were substantially shorter in non-stimulated or stimulated PRP than in PFP and PPP. This standardized PRP-CLT may become a useful tool to study the role of platelets in clot resistance and lysis. Our results suggest that initially, the platelets enmeshed in the clot slow down the fibrinolysis process. However, the increased clot resistance to lysis induced by TXA is overcome earlier in platelet-rich clots than in PFP or PPP clots. This is

  16. The effects of drugs used in anaesthesia on platelet membrane receptors and on platelet function.

    PubMed

    Kozek-Langenecker, Sibylle A

    2002-06-01

    Platelet dysfunctions are known origins of perioperative bleeding disorders which are a major concern in the management of surgical patients. Among multiple factors, interactions of drugs used in anaesthesia with platelets have been implicated to aggravate the risk of haemorrhagic complications. This paper reviews in vitro and in vivo studies which have examined the effects of inhalational, intravenous, and local anaesthetics, opioids, and muscle relaxants on platelets. A brief summary of platelet physiology, function tests, and flow cytometric assessment of membrane receptors is included. Although the results of many studies have been conflicting, it appears that halothane, sevoflurane, and propofol inhibit platelet function in a reversible and dose-related manner at concentrations used clinically. Ilalothane affects the intracellular activating second messenger inositol triphosphate, platelet calcium homeostasis, thromboxane A2 formation, and the inhibiting signal transduction pathway including cyclic adenosine monophosphate. The proposed platelet inhibiting mechanism of sevoflurane involves the suppression of thromboxane A2 formation. Propofol appears to cause platelet dysfunctions by inhibiting calcium mobilisation upon agonist stimulation. Nitrous oxide causes a modest suppression of calcium mobilisation. An interaction of local anaesthetics with components in the platelet membrane appears to account for their inhibiting effect, but only at concentrations far higher than that found during clinical use. A clinically relevant antithrombotic effect of regional anaesthesia has been observed, though. Isoflurane, enflurane, desflurane, barbiturates, etomidate, opioids, and muscle relaxants seem to have negligible effects on platelets at therapeutic concentrations. Anaesthetists should be aware of the potential impairment of the coagulation profile by anaesthetic agents.

  17. 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.

  18. Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates

    PubMed Central

    Koessler, Juergen; Weber, Katja; Koessler, Angela; Yilmaz, Pinar; Boeck, Markus; Kobsar, Anna

    2016-01-01

    Background The storage of platelets affects platelet integrity and functionality, a process named platelet storage lesion (PSL). Reduced adenosine diphosphate (ADP)-induced platelet aggregation is a typical manifestation of PSL. However, the role of ADP receptors in this context has not been evaluated yet. The aim of this study was, therefore, to investigate surface expression and function of the purinergic receptors P2Y1, P2Y12 and P2X1 in stored platelet concentrates. Material and methods Platelets were obtained from venous whole blood and from apheresis-derived platelet concentrates stored for 0, 2 and 5 days. Purinergic receptor expression was measured by flow cytometry and western blot analysis. Receptor function was determined by calcium-induced fluorescence (P2Y1 and P2X1) or by flow cytometric measurement of the platelet reactivity index (P2Y12). Results The basal surface expression and total content of purinergic receptors remained unchanged throughout storage. After an initial reduction during apheresis, P2X1-mediated calcium flux was maintained, whereas the P2Y1-mediated increase of calcium flux gradually decreased during the course of storage. In contrast, the platelet reactivity index was comparable in freshly obtained and stored platelets. Discussion The function of the P2Y12 receptor is maintained during storage of apheresis-derived platelet concentrates. However, the impairment of P2X1 and especially of P2Y1 receptor function indicated by decreased receptor-mediated calcium flux is an important mechanism contributing to reduced ADP responsiveness of stored platelets. PMID:26674810

  19. 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.

  20. 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.

  1. [Changes of platelet aggregation function of apheresis collected platelets and soluble P-selectin during storage].

    PubMed

    Xie, Zuo-Ting; Yang, Li-Hong; Tao, Zhi-Hua; Wang, Ming-Shan; Hong, Jun-Ying; Zhou, Wu; Chen, Zeng-Qiang; Dai, Mei-Jie

    2008-10-01

    The objective of this study was to explore the changes of aggregation function of apheresis platelets and soluble P-selectin (sP-selectin) during storage. 20 samples of apheresis platelets were collected, and the aggregation function were examined by function test and the level of sP-selectin every day in storage of 5 days. The results showed that the aggregation function of platelets declined obviously during storage, there were significant differences between the first-day group and any of the other groups (p < 0.01). The max platelet aggregation rate was < or = 3% in the fourth-day group; sP-selectin level in plasma increased with prolong of storage time; there were significant differences between the first-day group and any of the other groups (p < 0.05). In conclusion, platelets were activated continuously during storage, while its aggregation function declines significantly. The ability of platelet aggregation to response to ADP loses almost completely since the fourth day during platelet storage. It should be paid more attention to the damage of apheresis collected platelets during storage.

  2. Platelet function, activation and apoptosis during and after apheresis.

    PubMed

    Bakry, Rania; Sayed, Douaa; Galal, Hanan; Shaker, Sanaa

    2010-10-01

    Platelets are known to undergo shape change, activation, release reaction and apoptosis/necrosis during processing and storage. Apheresis may have a deleterious impact on platelet achievability and functional integrity. Platelet concentrates from 50 male volunteers obtained by COBE spectra were screened for platelet activation (CD62 and CD154) and apoptosis (phosphatidylserine detected by Annexin V). Donor samples before separation, during apheresis and at the third day of storage were used as baseline donor samples. Platelet aggregation to adenosine diphosphate (ADP) and collagen was performed. There was a statistically significant increase in the expression of activation markers in two different samples (during separation samples and third day samples). Although the increase in Annexin V expression was not so observable, it showed a significant increase also. There was marked decline in the platelet aggregation. The correlations between the values of CD62, CD154 and Annexin V were detected in baseline samples and increased during separation and at the third day of platelets storage. Correlation between values of platelet aggregation to collagen and Annexin V was relevant only in the baseline samples. No other correlations were encountered between platelet aggregation and markers of activation and apoptosis during apheresis and storage. Initial platelet activation induced by apheresis may have an impact on phosphatidylserine expression with no impact on aggregation function of platelets during storage.

  3. 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

  4. Transporters in human platelets: physiologic function and impact for pharmacotherapy.

    PubMed

    Jedlitschky, Gabriele; Greinacher, Andreas; Kroemer, Heyo K

    2012-04-12

    Platelets store signaling molecules (eg, serotonin and ADP) within their granules. Transporters mediate accumulation of these molecules in platelet granules and, on platelet activation, their translocation across the plasma membrane. The balance between transporter-mediated uptake and elimination of signaling molecules and drugs in platelets determines their intracellular concentrations and effects. Several members of the 2 major transporter families, ATP-binding cassette (ABC) transporters and solute carriers (SLCs), have been identified in platelets. An example of an ABC transporter is MRP4 (ABCC4), which facilitates ADP accumulation in dense granules. MRP4 is a versatile transporter, and various additional functions have been proposed, notably lipid mediator release and a role in aspirin resistance. Several other ABC proteins have been detected in platelets with functions in glutathione and lipid homeostasis. The serotonin transporter (SERT, SLC6A4) in the platelet plasma membrane represents a well-characterized example of the SLC family. Moreover, recent experiments indicate expression of OATP2B1 (SLCO2B1), a high affinity transporter for certain statins, in platelets. Changes in transporter localization and expression can affect platelet function and drug sensitivity. This review summarizes available data on the physiologic and pharmacologic role of transporters in platelets.

  5. FXR and its ligands inhibit the function of platelets

    PubMed Central

    Vaiyapuri, Sakthivel; Ali, Marfoua S.; Sasikumar, Parvathy; Sage, Tanya; Flora, Gagan D; Bye, Alex P; Kriek, Neline; Dorchies, Emilie; Molendi-Coste, Olivier; Dombrowicz, David; Staels, Bart; Bishop-Bailey, David

    2016-01-01

    Objective While initially seemingly paradoxical due to the lack of nucleus, platelets possess a number of transcription factors that regulate their function through DNA-independent mechanisms. These include the Farnesoid X Receptor (FXR), a member of the superfamily of ligand-activated transcription factors that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6-ECDCA, modulate platelet activation nongenomically. Approach and Results FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization and secretion, fibrinogen binding and aggregation. Exposure to FXR ligands also reduced integrin αIIbβ3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cGMP levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the non-genomic actions of these ligands to the FXR receptor. Conclusion This study provides support for the ability of FXR ligands to modulate platelet activation. The athero-protective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for prevention of athero-thrombotic disease. PMID:27758768

  6. 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

  7. Functional Assays for Ricin Detection

    NASA Astrophysics Data System (ADS)

    Ezan, Eric; Duriez, Elodie; Fenaille, François; Becher, François

    In this review, we provide background information on ricin structure, present available functional assays for other toxins that are potential biothreat agents, and finish by describing the functional assay of ricin itself. Using appropriate sample preparation and optimized detection based on N-glycosidase activity, we demonstrate that specific detection of whole ricin at a level of around 0.1 ng/mL is possible and applicable to environmental samples.

  8. The functional role of platelets in the regulation of angiogenesis.

    PubMed

    Walsh, Tony G; Metharom, Pat; Berndt, Michael C

    2015-01-01

    Functionally, platelets are primarily recognized as key regulators of thrombosis and hemostasis. Upon vessel injury, the typically quiescent platelet interacts with subendothelial matrix to regulate platelet adhesion, activation and aggregation, with subsequent induction of the coagulation cascade forming a thrombus. Recently, however, newly described roles for platelets in the regulation of angiogenesis have emerged. Platelets possess an armory of pro- and anti-angiogenic proteins, which are actively sequestered and highly organized in α-granule populations. Platelet activation facilitates their release, eliciting potent angiogenic responses through mechanisms that appear to be tightly regulated. In conjunction, the release of platelet-derived phospholipids and microparticles has also earned merit as synergistic regulators of angiogenesis. Consequently, platelets have been functionally implicated in a range of angiogenesis-dependent processes, including physiological roles in wound healing, vascular development and blood/lymphatic vessel separation, whilst facilitating aberrant angiogenesis in a range of diseases including cancer, atherosclerosis and diabetic retinopathy. Whilst the underlying mechanisms are only starting to be elucidated, significant insights have been established, suggesting that platelets represent a promising therapeutic strategy in diseases requiring angiogenic modulation. Moreover, anti-platelet therapies targeting thrombotic complications also exert protective effects in disorders characterized by persistent angiogenesis.

  9. 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

  10. Metastasis: new functional implications of platelets and megakaryocytes.

    PubMed

    Leblanc, Raphael; Peyruchaud, Olivier

    2016-07-07

    Platelets are essential components of hemostasis. Due to a plethora of factors released on activation, platelet functions are also connected to tumor growth, notably by acting on angiogenesis. It is now well recognized that major roles of platelets in the poor outcome of cancer patients occurs during hematogenous dissemination of cancer cells. In this review, we describe recent insights into the molecular mechanisms supporting the prometastatic activity of platelets. Platelets have been shown to promote survival of circulating tumor cells (CTCs) in the bloodstream by conferring resistance to the shear stress and attack from natural killer cells. Recently, platelets were found to promote and/or maintain the state of epithelial to mesenchymal transition on CTCs through platelet secretion of transforming growth factor β in response to CTC activation. At a later stage in the metastatic process, platelets promote extravasation and establishment of metastatic cells in distant organs as observed in bone. This particular environment is also the site of hematopoiesis, megakaryocytopoiesis, and platelet production. Increasing the number of megakaryocytes (MKs) in the bone marrow results in a high bone mass phenotype and inhibits skeletal metastasis formation of prostate cancer cells. As a result of their specific location in vascular niches in the bone marrow, MK activity might contribute to the "seed and soil" suitability between CTCs and bone. In conclusion, recent findings have made a great advance in our knowledge on how platelets contribute to the metastatic dissemination of cancer cells and that may support the development of new antimetastasis therapies.

  11. Technical Advance: Changes in neutrophil migration patterns upon contact with platelets in a microfluidic assay.

    PubMed

    Frydman, Galit H; Le, Anna; Ellett, Felix; Jorgensen, Julianne; Fox, James G; Tompkins, Ronald G; Irimia, Daniel

    2017-03-01

    Neutrophils are traditionally regarded as the "first responders" of the immune system. However, recent observations revealed that platelets often respond earlier to recruit and activate neutrophils within sites of injury and inflammation. Currently, platelet-neutrophil interactions are studied by intravital microscopy. Although such studies provide exceptional, physiologic in vivo data, they are also laborious and have low throughput. To accelerate platelet-neutrophil interaction studies, we have developed and optimized an ex vivo microfluidic platform with which the interactions between platelets and moving neutrophils are measured at single-cell level in precise conditions and with high throughput. With the use of this new assay, we have evaluated changes in neutrophil motility upon direct contact with platelets. Motility changes include longer distances traveled, frequent changes in direction, and faster neutrophil velocities compared with a standard motility response to chemoattractant fMLP. We also found that the neutrophil-platelet direct interactions are transient and mediated by CD62P-CD162 interactions, localized predominantly at the uropod of moving neutrophils. This "crawling," oscillatory neutrophil behavior upon platelet contact is consistent with previous in vivo studies and validates the use of this new test for the exploration of this interactive relationship.

  12. Reversible translocation of glycoprotein Ib in plasmin-treated platelets: consequences for platelet function.

    PubMed

    Lu, H; Soria, C; Soria, J; De Romeuf, C; Perrot, J Y; Tenza, D; Garcia, I; Caen, J P; Cramer, E M

    1993-12-01

    Understanding the effect of fibrinolysis on platelet function is of clinical importance. Plasmin is recognized to affect platelet adhesive function by reducing the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor (vWF) bound to the subendothelium. This platelet function is commonly explored in vitro by the ristocetin-induced agglutination test. Our previous study demonstrated a plasmin-induced redistribution of GP Ib molecules from the platelet surface to the linings of the surface-connected canalicular system (SCCS), a critical mechanism for understanding plasmin-induced GP Ib dysfunction. Here, we demonstrate that neutralization of plasmin by its inhibitors, aprotinin or tripeptide Val-Phe-Lys-CH2Cl, permits a time dependent recovery (within 30 min) of ristocetin-induced agglutination in the platelets which were stimulated by plasmin at < 1 CU ml-1. This functional recovery was accompanied with a restoration of a normal amount of GP Ib on the platelet surface, as measured by the binding of both monoclonal anti-GP Ib antibody SZ 2 and 125I-labelled vWF to the platelets. Cytochalasin D did not inhibit this recovery, suggesting that this process may be due to passive actin depolymerization. These findings were further confirmed by immunoelectron microscopic study. Utilizing the platelets pre-labelled with anti-GP Ib antibody prior to plasmin stimulation, it was demonstrated that the observed recovery is due to a reverse translocation from the SCCS to the plasma membrane of the same GP Ib molecules which were present initially at the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. IVBT-documented platelet function correlates with flow cytometric data.

    PubMed

    Hoffmann, J; Bonacker, G; Kretschmer, V; Schulzki, T; Heimanns, J

    1996-12-01

    Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (fibrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate time (ST). The thrombocytes with the most impaired function (OT > or = 485 s/ST > 30 min) had significantly less platelet fluorescence when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT < 100 s/ST < 15 min). Similar results were obtained when evaluating the data relative to the bone marrow status: patients with < 1000 WBC/microliters showed significantly less platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets/microliters a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the

  14. Generation of functional platelets from canine induced pluripotent stem cells.

    PubMed

    Nishimura, Toshiya; Hatoya, Shingo; Kanegi, Ryoji; Sugiura, Kikuya; Wijewardana, Viskam; Kuwamura, Mitsuru; Tanaka, Miyuu; Yamate, Jyoji; Izawa, Takeshi; Takahashi, Masahiro; Kawate, Noritoshi; Tamada, Hiromichi; Imai, Hiroshi; Inaba, Toshio

    2013-07-15

    Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

  15. Ultraviolet irradiation of platelet concentrate abrogates lymphocyte activation without affecting platelet function in vitro

    SciTech Connect

    Kahn, R.A.; Duffy, B.F.; Rodey, G.G.

    1985-11-01

    We studied the effect of ultraviolet (UV) radiation on platelet concentrates. Samples irradiated at 310 mm for 30 minutes at a dose of 1782 J per m2 showed no loss of platelet function in vitro as determined by adenosine diphosphate, collagen, or ristocetin-induced aggregation. Lymphocytes isolated from irradiated units were unable to act as responders or stimulators in a mixed-lymphocyte reaction. These data suggest that UV radiation of platelet concentrates may result in a cell suspension that is unable to evoke an immunological response.

  16. Alterations of platelet function and clot formation kinetics following in vitro exposure to anti-A and -B antibodies

    PubMed Central

    Refaai, Majed A.; Carter, Jessie; Henrichs, Kelly F.; Davidson, Donna C.; Pollock, Stephen J.; Casey, Ann E.; Spinelli, Sherry L.; Phipps, Richard P.; Francis, Charles W.; Blumberg, Neil

    2012-01-01

    Background ABO mismatched platelets are commonly transfused despite reported complications. We hypothesized that because platelets possess A and B antigens on their surface, ABO mismatched transfused or recipient platelets could become activated and/or dysfunctional after exposure to anti-A or -B antibodies in the transfused or recipient plasma. We present here in vitro modeling data on the functional effects of exposure of platelets to ABO antibodies. Methods Platelet functions of normal platelets of all ABO types were assessed before and after incubation with normal saline, ABO identical plasmas, or O plasmas with varying titers of anti-A and anti-B (anti-A/B) antibodies. Assays used for this assessment include: platelet aggregation, clot kinetics, thrombin generation, platelet cytoskeletal function, and mediator release. Results Exposure of antigen bearing platelets to O plasma with moderate to high titers of anti-A/B antibodies significantly inhibits aggregation, prolongs PFA-100 epinephrine closure time, disrupts clot formation kinetics, accelerates thrombin generation, reduces total thrombin production, alters platelet cytoskeletal function, and influences pro-inflammatory and pro-thrombotic mediator release. Conclusions Our findings demonstrate a wide range of effects that anti-A/B antibodies have on platelet function, clot formation, thrombin generation, platelet cytoskeletal function, and mediator release. These data provide potential explanations for clinical observations of increased red cell utilization in trauma and surgical patients receiving ABO non-identical blood products. Impaired hemostasis caused by anti-A/B antibodies interacting with A and B antigens on platelets, soluble proteins, and perhaps even endothelial cells is a potential contributing factor to hemorrhage in patients receiving larger volumes of ABO non-identical transfusions. PMID:22624532

  17. Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice.

    PubMed

    Lu, Shi-Jiang; Li, Feng; Yin, Hong; Feng, Qiang; Kimbrel, Erin A; Hahm, Eunsil; Thon, Jonathan N; Wang, Wei; Italiano, Joseph E; Cho, Jaehyung; Lanza, Robert

    2011-03-01

    Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

  18. Morphological and functional platelet abnormalities in Berkeley sickle cell mice.

    PubMed

    Shet, Arun S; Hoffmann, Thomas J; Jirouskova, Marketa; Janczak, Christin A; Stevens, Jacqueline R M; Adamson, Adewole; Mohandas, Narla; Manci, Elizabeth A; Cynober, Therese; Coller, Barry S

    2008-01-01

    Berkeley sickle cell mice are used as animal models of human sickle cell disease but there are no reports of platelet studies in this model. Since humans with sickle cell disease have platelet abnormalities, we studied platelet morphology and function in Berkeley mice (SS). We observed elevated mean platelet forward angle light scatter (FSC) values (an indirect measure of platelet volume) in SS compared to wild type (WT) (37+/-3.2 vs. 27+/-1.4, mean+/-SD; p<0.001), in association with moderate thrombocytopenia (505+/-49 x 10(3)/microl vs. 1151+/-162 x 10(3)/microl; p<0.001). Despite having marked splenomegaly, SS mice had elevated levels of Howell-Jolly bodies and "pocked" erythrocytes (p<0.001 for both) suggesting splenic dysfunction. SS mice also had elevated numbers of thiazole orange positive platelets (5+/-1% vs. 1+/-1%; p<0.001), normal to low plasma thrombopoietin levels, normal plasma glycocalicin levels, normal levels of platelet recovery, and near normal platelet life spans. Platelets from SS mice bound more fibrinogen and antibody to P-selectin following activation with a threshold concentration of a protease activated receptor (PAR)-4 peptide compared to WT mice. Enlarged platelets are associated with a predisposition to arterial thrombosis in humans and some humans with SCD have been reported to have large platelets. Thus, additional studies are needed to assess whether large platelets contribute either to pulmonary hypertension or the large vessel arterial occlusion that produces stroke in some children with sickle cell disease.

  19. Platelet membrane glycoproteins and their function: an overview.

    PubMed

    Kunicki, T J

    1989-07-01

    The membrane glycoproteins (GP) of human platelets act as receptors that mediate two important functions, adhesion to the subendothelial matrix and platelet-platelet cohesion, or aggregation. Many of these glycoprotein receptors exist as noncovalently linked heterodimers, including those that belong to the supergene family of adhesion receptors called the integrins. Human platelets contain at least five members of this integrin family, including a collagen receptor (GP Ia-IIa; alpha 2, beta 1), a fibronectin receptor (GP Ic-IIa; alpha 5, beta 1), a laminin receptor (GP Ic'-IIa; alpha 6, beta 1), a vitronectin receptor (VnR; alpha v, beta 3), and a promiscuous, activation-dependent receptor that is thought to be the receptor most responsible for fibrinogen-dependent, platelet-platelet cohesion (GP IIb-IIIa; alpha IIb, beta 3). Some, but not all, of the integrins bind to a tripeptide sequence, arginine-glycine-aspartic acid (RGD), on the adhesive proteins. In addition to the integrins, platelets contain other membrane glyco-proteins: GP Ib-IX, a receptor for von Willebrand factor, which is thought to be the receptor most responsible for platelet adhesion to the subendothelial matrix in a flowing system; GP V, which may be associated with GP Ib-IX and whose function remains unknown; and GP IV (GP IIIb), which functions as a receptor for thrombospondin and collagen.

  20. 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

  1. Role of reactive nitrogen species in blood platelet functions.

    PubMed

    Olas, Beata; Wachowicz, Barbara

    2007-12-01

    Blood platelets, in analogy to other circulating blood cells, can generate reactive oxygen/nitrogen species (ROS/RNS) that may behave as second messengers and may regulate platelet functions. Accumulating evidence suggest a role of ROS/RNS in platelet activation. On the other hand, an increased production of ROS/RNS causes oxidative stress, and thus, may contribute to the development of different diseases, including vascular complications, inflammatory and psychiatric illnesses. Oxidative stress in platelets leads to chemical changes in a wide range of their components, and platelet proteins may be initial targets of ROS/RNS action. It has been demonstrated that reaction of proteins with ROS/RNS results in the oxidation and nitration of some amino acid residues, formation of aggregates or fragmentation of proteins. In oxidized proteins new carbonyl groups and protein hydroperoxides are also formed. In platelets, low molecular weight thiols such as glutathione (GSH), cysteine and cysteinylglycine and protein thiols may be also target for ROS/RNS action. This review describes the chemical structure and biological activities of reactive nitrogen species, mainly nitric oxide ((*)NO) and peroxynitrite (ONOO(-)) and their effects on blood platelet functions, and the mechanisms involved in their action on platelets.

  2. Impact of high dose vitamin C on platelet function

    PubMed Central

    Mohammed, Bassem M; Sanford, Kimberly W; Fisher, Bernard J; Martin, Erika J; Contaifer Jr, Daniel; Warncke, Urszula Osinska; Wijesinghe, Dayanjan S; Chalfant, Charles E; Brophy, Donald F; Fowler III, Alpha A; Natarajan, Ramesh

    2017-01-01

    AIM To examine the effect of high doses of vitamin C (VitC) on ex vivo human platelets (PLTs). METHODS Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide. The doses of VitC used here correspond to plasma VitC levels reported in recently completed clinical trials. Prior to supplementation, a baseline sample was collected for analysis. PLTs were sampled again on days 2, 5 and 8 and assayed for changes in PLT function by: Thromboelastography (TEG), for changes in viscoelastic properties; aggregometry, for PLT aggregation and adenosine triphosphate (ATP) secretion in response to collagen or adenosine diphosphate (ADP); and flow cytometry, for changes in expression of CD-31, CD41a, CD62p and CD63. In addition, PLT intracellular VitC content was measured using a fluorimetric assay for ascorbic acid and PLT poor plasma was used for plasma coagulation tests [prothrombin time (PT), partial thrombplastin time (PTT), functional fibrinogen] and Lipidomics analysis (UPLC ESI-MS/MS). RESULTS VitC supplementation significantly increased PLTs intracellular ascorbic acid levels from 1.2 mmol/L at baseline to 3.2 mmol/L (Lo VitC) and 15.7 mmol/L (Hi VitC, P < 0.05). VitC supplementation did not significantly change PT and PTT values, or functional fibrinogen levels over the 8 d exposure period (P > 0.05). PLT function assayed by TEG, aggregometry and flow cytometry was not significantly altered by Lo or Hi VitC for up to 5 d. However, PLTs exposed to 3 mmol/L VitC for 8 d demonstrated significantly increased R and K times by TEG and a decrease in the α-angle (P < 0.05). There was also a fall of 20 mm in maximum amplitude associated with the Hi VitC compared to

  3. 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.

  4. Platelet function and fibrinolytic activity following distance running.

    PubMed

    Knudsen, J B; Brodthagen, U; Gormsen, J; Jordal, R; Nørregaard-Hansen, K; Paulev, P E

    1982-11-01

    6 long distance runners from the Danish marathon elite and 6 non-runners completed test runs of 28 and 12 km, respectively. Distance runners and non-runners showed the same responses in platelet function. We found a significant decrease in ADP induced platelet aggregability, a decreased serotonin release induced by ADP and collagen and an increase in platelet factor 4 immediately following the run. The antithrombin III levels remained constant. Euglobulin lysis time was shortened (by approximately 50%) and the plasminogen levels significantly increased. The last 2 findings indicate an equal increase in fibrinolytic activity during distance running in both groups. While short term, strenuous exercise induces platelet hyperaggregation, long term distance running induces a state of exhaustion of platelet aggregation capacity.

  5. Which platelet function test best reflects the in vivo plasma concentrations of ticagrelor and its active metabolite? The HARMONIC study.

    PubMed

    Koziński, Marek; Ostrowska, Małgorzata; Adamski, Piotr; Sikora, Joanna; Sikora, Adam; Karczmarska-Wódzka, Aleksandra; Marszałł, Michał Piotr; Boinska, Joanna; Laskowska, Ewa; Obońska, Ewa; Fabiszak, Tomasz; Kubica, Jacek

    2016-11-30

    Aim of this study was assessment of the relationship between concentrations of ticagrelor and its active metabolite (AR-C124910XX) and results of selected platelet function tests. In a single-centre, cohort study, patients with myocardial infarction underwent blood sampling following a 180 mg ticagrelor loading dose intake (predose, 1, 2, 3, 4, 6, 12, 24 hours postdose) to perform pharmacokinetic and pharmacodynamic assessments. Platelet reactivity was evaluated using the VASP-assay, the VerifyNow device and the Multiplate analyzer. Analysis of 36 patients revealed high negative correlations between ticagrelor concentrations and platelet reactivity evaluated with all three platelet function tests (the VASP-assay: RS=-0.722; p<0.0001; the VerifyNow device: RS=-0.715; p<0.0001; the Multiplate analyzer: RS=-0.722; p<0.0001), with no significant differences between correlation coefficients. Similar results were found for AR-C124910XX. Platelet reactivity values assessed with all three methods generally correlated well with each other; however, a significantly higher correlation (p<0.02) was demonstrated between the VerifyNow and Multiplate tests (RS=0.707; p<0.0001) than in other assay combinations (the VASP-assay and the VerifyNow device: RS=0.595; p<0.0001; the VASP-assay and the Multiplate analyzer: RS=0.588; p<0.0001). With respect to the recognition of high platelet reactivity, we found higher measurement concordance between the VerifyNow and Multiplate tests compared with other assay combinations, while for low platelet reactivity, only results of the VerifyNow and Multiplate assay were related to each other. Platelet reactivity measurements performed with the VASP, VerifyNow and Multiplate tests show comparably strong negative correlations with ticagrelor and AR-C124910XX concentrations.

  6. [Acquired platelet function disorders: pathogenesis, classification, frequency, diagnosis, clinical management].

    PubMed

    Scharf, R E

    2008-12-01

    Given the high consumption of pharmacological agents in western societies, it is not surprising at all that drugs represent the most common cause of acquired platelet dysfunction. While acetylsalicylic acid, clopigogrel and integrin alphaIIbbeta3 (GPIIb-IIIa) receptor antagonists are well-known as prototypes of antiplatelet drugs, other widely used agents including non-steroidal anti-inflammatory drugs, antibiotics, serotonin reuptake inhibitors, and volume expanders can also impair platelet function and cause or aggravate haemorrhages. Besides pharmacological agents, certain clinical conditions are often associated with qualitative platelet disorders and bleeding diathesis. Consequently, in contrast to inherited platelet disorders, acquired platelet function defects are much more frequent in clinical practice and deserve special attention. Their pathogenesis is widespread and heterogeneous with various, sometimes overlapping abnormalities. Moreover, acquired platelet dysfunctions can occur at any age and range in severity from mild to life-threatening haemorrhages. Due to their heterogeneity, acquired platelet function disorders will be classified and discussed according to the underlying clinical setting or disease.

  7. Platelet-derived microparticles and platelet function profile in children with congenital heart disease.

    PubMed

    Ismail, Eman Abdel Rahman; Youssef, Omneya Ibrahim

    2013-01-01

    Platelet microparticles (PMPs) and function profile in children with congenital heart disease (CHD) have not been widely explored. We investigated platelet aggregation, flow cytometric platelet surface receptors (P-selectin and glycoprotein (GP) IIb/IIIa) and PMPs in 23 children with cyanotic CHD (CCHD), 30 children with acyanotic CHD (ACHD) and 30 healthy controls correlating these variables to hematological and coagulation parameters including von Willebrand factor antigen (vWF Ag) as a marker of endothelial dysfunction. Hemoglobin, hematocrit (HCT), D-dimer, and vWF Ag were significantly higher in CCHD than ACHD group. Platelet MPs and P-selectin expression were increased in patients than controls, particularly in CCHD and positively correlated to HCT, D-dimer, and vWF Ag while platelet count, aggregation, and GP IIb/IIIa expression were decreased in CCHD compared with ACHD group and negatively correlated to HCT. The overproduction of PMPs and platelet activation with suppressed aggregation may be implicated in the pathogenesis of coagulation/hemostatic abnormalities in children with CCHD.

  8. Risk factors for coronary heart disease and platelet functions.

    PubMed

    Renaud, S

    1984-01-01

    Epidemiologic studies have shown that several environmental factors are associated with coronary heart disease (CHD). Most of them are predisposing factors known also as risk factors. Other factors appear to have preventive effects. Blood lipids have been considered the main blood mediator between most of these factors and CHD. In recent years, this concept has been challenged since many of these factors did not affect serum lipids. By contrast blood platelets, involved in both thrombosis and atherosclerosis, appear to have their functions markedly changed by most of the factors associated with CHD. To determine whether saturated fats would affect platelet functions as shown in animals and in pilot studies in man, groups of male farmers (40-45 years) from 2 regions of France (Var and Moselle) in which the mortality rate from CHD differed markedly were studied, particularly regarding their platelet functions in relation to the intake of saturated fats. No difference could be observed in blood between the 2 regions concerning total cholesterol, HDL cholesterol, or triglycerides, the coagulation was markedly accelerated, as well as the platelet clotting activity in farmers from Moselle. The response of platelets mostly to thrombin but also to adenosine diphosphate (ADP), epinephrine, and collagen was more elevated in Moselle farmers. In Moselle farmers, there was significantly higher intake of saturated fats (16% of the calories) as compared to Var (12%). To determine whether the abnormal platelet response in Moselle farmers was really due to the diet or whether a genetic factor might be involved, a group of 50 Moselle farmers were persuaded to change their dietary habits in order to lower their intake of saturated fats to 10% of the calories and that of polyunsaturated to approximately 12%. 1 year after diet modification, the clotting time (PCT) and clotting activity of platelets were considerably prolonged and the response to thrombin drastically reduced. These

  9. 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.

  10. 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

    2012-07-05

    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.

  11. 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.

  12. Platelet functional and transcriptional changes induced by intralipid infusion.

    PubMed

    Beaulieu, Lea M; Vitseva, Olga; Tanriverdi, Kahraman; Kucukural, Alper; Mick, Eric; Hamburg, Naomi; Vita, Joseph; Freedman, Jane E

    2016-06-02

    Multiple studies have shown the effects of long-term exposure to high-fat or western diets on the vascular system. There is limited knowledge on the acute effects of high circulating fat levels, specifically on platelets, which have a role in many processes, including thrombosis and inflammation. This study investigated the effects of acute, high-fat exposure on platelet function and transcript profile. Twenty healthy participants were given an intravenous infusion of 20% Intralipid emulsion and heparin over 6 hours. Blood samples were taken prior to and the day after infusion to measure platelet function and transcript expression levels. Platelet aggregation was not significantly affected by Intralipid infusion, but, when mitochondria function was inhibited by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin, platelet aggregation was higher in the post-infusion state compared to baseline. Through RNA sequencing, and verified by RT-qPCR, 902 miRNAs and 617 mRNAs were affected by Intralipid infusion. MicroRNAs increased include miR-4259 and miR-346, while miR-517b and miR-517c are both decreased. Pathway analysis identified two clusters significantly enriched, including cell motility. In conclusion, acute exposure to high fat affects mitochondrial-dependent platelet function, as well as the transcript profile.

  13. Effect on platelet functions of derivatives of cyclic nucleotides.

    PubMed

    Pareti, F I; Carrera, D; Mannucci, L; Mannucci, P M

    1978-04-30

    Derivatives of cyclic nucleotides were evaluated for their ability to inhibit platelet aggregation and the release reaction. Derivatives substituted in position 8 (mainly 8-Br-cyclic GMP) were more active than 3'-5' cyclic AMP, and their relative potency in inhibiting platelet aggregation and 14C-serotonin release was comparable to that of N62-0'-dibutyryl-cyclic AMP. Compounds substituted in position 6 or 2'-0 were not effective. The active compounds, which were also tested for their ability to stimulate platelet adenylate cyclase or to inhibit cyclic AMP phosphodiesterase, did not modify the intracellular levels of cyclic AMP. Since previous animal experiments have shown that these derivatives cause less side effects than cyclic AMP and its dibutyryl derivative in animals, it is suggested that modification of the cyclophosphate molecule might make it possible to find compounds active only on platelet function without interfering with other biological systems.

  14. 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.

  15. Influence of gold nanoparticles on platelets functional activity in vitro

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Akchurin, George G.; Ivanov, Alexey N.; Kirichuk, Vyacheslav F.; Terentyuk, George S.; Khlebtsov, Boris N.; Khlebtsov, Nikolay G.

    2008-02-01

    Now in the leading biomedical centers of the world approved new technology of laser photothermal destruction of cancer cells using plasmon gold nanoparticles. Investigations of influence of gold nanoparticles on white rat platelets aggregative activity in vitro have been made. Platelet aggregation was investigated in platelet rich plasma (PRP) with help of laser analyzer 230 LA <>, Russia). Aggregation inductor was ADP solution in terminal concentration 2.5 micromole (<>, Russia). Gold nanoshells soluted in salt solution were used for experiments. Samples of PRP were incubated with 50 or 100 μl gold nanoshells solution in 5 minute, after that we made definition ADP induced platelet aggregation. We found out increase platelet function activity after incubation with nanoparticles solution which shown in maximum ADP-induced aggregation degree increase. Increase platelet function activity during intravenous nanoshells injection can be cause of thrombosis on patients. That's why before clinical application of cancer cell destruction based on laser photothermal used with plasmon gold nanoparticles careful investigations of thrombosis process and detail analyze of physiological blood parameters are very necessary.

  16. A simple colorimetric assay for phenotyping the major human thermostable phenol sulfotransferase (SULT1A1) using platelet cytosols.

    PubMed

    Frame, L T; Ozawa, S; Nowell, S A; Chou, H C; DeLongchamp, R R; Doerge, D R; Lang, N P; Kadlubar, F F

    2000-09-01

    A thermostable phenol sulfotransferase, SULT1A1, has been implicated in numerous detoxification and bioactivation pathways; however, little is known regarding its endogenous function or its putative role in mediating risk for human environmental disease. A simple endpoint colorimetric assay is described that can be used for rapid phenotyping of SULT1A1 activity in human populations. The assay utilizes a microtiter-plate format and relatively small amounts of platelet cytosol-derived enzyme. The enzyme catalyzes the synthesis of 2-naphthylsulfate from 2-naphthol and 5'-phosphoadenosine 3'-phosphosulfate (PAPS), whereas addition of p-nitrophenyl sulfate to the assay contributes to an effective PAPS-regenerating system. In contrast to other sulfotransferase assay methods, 3'-phosphoadenosine 5'-phosphate (PAP) does not accumulate during the incubation to interfere with enzyme activity, but instead serves as a cofactor to cause the removal of sulfate from p-nitrophenyl sulfate to regenerate PAPS. This reaction concomitantly results in generation of p-nitrophenol that can be quantified colorimetrically at 405 nm (epsilon = 18,200 M(-1)) to give an indirect measure of sulfotransferase activity. Using platelet enzyme preparations from adult human subjects, sulfation rates of two prototypical thermostable phenol sulfotransferase substrates (2-naphthol and p-nitrophenol) and one thermolabile phenol sulfotransferase substrate (dopamine) were determined using standard radiochemical protocols. These data were then compared with results from the colorimetric assay using 2-naphthol as substrate. There was a good correlation between the phenotyping assay and radiochemical assays for both 2-naphthol sulfotransferase and p-nitrophenol sulfotransferase activity (r = 0.85 and 0.69, respectively). However, SULT1A1 activity was approximately 10 to 20 times higher with the colorimetric determination. As anticipated, there was no correlation between SULT1A1 activity and dopamine

  17. 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

  18. 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

  19. 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.

  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. Functional Effect of the Mutations Similar to the Cleavage during Platelet Activation at Integrin β3 Cytoplasmic Tail when Expressed in Mouse Platelets

    PubMed Central

    Huang, Jiansong; Long, Zhangbiao; Zhou, Yulan; Liu, Ping; Tao, Lanlan; Ruan, Zheng; Xiao, Bing; Zhang, Wei; Li, Dongya; Dai, Kesheng; Mao, Jianhua; Xi, Xiaodong

    2016-01-01

    Previous studies in Chinese hamster ovary cells showed that truncational mutations of β3 at sites of F754 and Y759 mimicking calpain cleavage regulate integrin signaling. The roles of the sequence from F754 to C-terminus and the conservative N756ITY759 motif in platelet function have yet to be elaborated. Mice expressing β3 with F754 and Y759 truncations, or NITY deletion (β3-ΔTNITYRGT, β3-ΔRGT, or β3-ΔNITY) were established through transplanting the homozygous β3-deficient mouse bone marrow cells infected by the GFP tagged MSCV MigR1 retroviral vector encoding different β3 mutants into lethally radiated wild-type mice. The platelets were harvested for soluble fibrinogen binding and platelet spreading on immobilized fibrinogen. Platelet adhesion on fibrinogen- and collagen-coated surface under flow was also tested to assess the ability of the platelets to resist hydrodynamic drag forces. Data showed a drastic inhibition of the β3-ΔTNITYRGT platelets to bind soluble fibrinogen and spread on immobilized fibrinogen in contrast to a partially impaired fibrinogen binding and an almost unaffected spreading exhibited in the β3-ΔNITY platelets. Behaviors of the β3-ΔRGT platelets were consistent with the previous observations in the β3-ΔRGT knock-in platelets. The adhesion impairment of platelets with the β3 mutants under flow was in different orders of magnitude shown as: β3-ΔTNITYRGT>β3-ΔRGT>β3-ΔNITY to fibrinogen-coated surface, and β3-ΔTNITYRGT>β3-ΔNITY>β3-ΔRGT to collagen-coated surface. To evaluate the interaction of the β3 mutants with signaling molecules, GST pull-down and immunofluorescent assays were performed. Results showed that β3-ΔRGT interacted with kindlin but not c-Src, β3-ΔNITY interacted with c-Src but not kindlin, while β3-ΔTNITYRGT did not interact with both proteins. This study provided evidence in platelets at both static and flow conditions that the calpain cleavage-related sequences of integrin β3, i.e. T755

  2. Resveratrol, a phenolic antioxidant with effects on blood platelet functions.

    PubMed

    Olas, Beata; Wachowicz, Barbara

    2005-08-01

    The main purpose of this article is to provide an overview of the currently available evidence of antiplatelet properties of resveratrol (3,4('),5-trihydroxystilbene). Resveratrol, a phenolic compound found naturally in fruits, nuts, flowers, seeds and bark of different plants is integral part of human diet. It exhibits a wide range of biological effects, including antiplatelet, anti-inflammatory, anticancer, antimutagenic and antifungal properties. It is also a potent antioxidant, reactive oxygen species scavenger and metal chelators. Resveratrol reduces lipid peroxidation, oxidation and nitration of platelet and plasma proteins. This review article describes the chemical structure of resveratrol, its biological activity, the effects on blood platelet functions and the mechanisms involved in its action on blood platelets, the cells which play an important role not only in the haemostatic process, but also in pathogenesis of cardiovascular diseases.

  3. Comparison of modified Thrombelastograph and Plateletworks whole blood assays to optical platelet aggregation for monitoring reversal of clopidogrel inhibition in elective surgery patients.

    PubMed

    Craft, Robert M; Chavez, Jack J; Snider, Carolyn C; Muenchen, Robert A; Carroll, Roger C

    2005-06-01

    Clinically monitoring recovery from clopidogrel and nonsteroidal anti-inflammatory drug (NSAID) inhibition requires whole blood assays corresponding to a standard methodology such as platelet-rich plasma aggregation monitored optically (OPA). We compared OPA, using an ED 50 dose of adenosine diphosphate activation, with 2 whole blood assays, Plateletworks (PWA) and modified Thrombelastograph (TEG). Two sets of assays were performed on 43 surgery patients while on clopidogrel and off clopidogrel to determine the reversal of absolute and relative inhibition. The modified TEG had Spearman correlations with OPA for absolute (rho = .424; P = .006) and relative inhibition (rho = .742; P < .0001). PWA correlations with OPA gave absolute (rho = .28; P = .08) and relative inhibition (rho = .46; P = .004) values. Bland-Altman analysis indicated agreement of both tests with OPA, showing constant biases of about 18% and some dependency on mean magnitude error. Cohen effect size thresholds defined nonresponders as < 7.7% clopidogrel inhibition relative to baseline recovery of full platelet function. Apparent nonresponse to clopidogrel or lack of platelet recovery did not correlate with statin or NSAID therapies. These PWA and modified TEG whole blood assays could prove useful for monitoring the reversal of clopidogrel and NSAID inhibition before surgery. More important, these assays done at baseline and after beginning clopidogrel therapy could monitor the effectiveness for the individual patients with cardiovascular disease and help identify the need for alternative therapies.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen + ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin + UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established. PMID:24749844

  6. Increasing platelet concentration in platelet-rich plasma inhibits anterior cruciate ligament cell function in three-dimensional culture.

    PubMed

    Yoshida, Ryu; Cheng, Mingyu; Murray, Martha M

    2014-02-01

    Tissue engineering is one new strategy being developed to treat ACL ruptures. One such approach is bio-enhanced ACL repair, where a suture repair is supplemented with a bio-active scaffold containing platelets. However, the optimal concentration of platelets to stimulate ACL healing is not known. We hypothesized that increasing platelet concentrations in the scaffold would enhance critical cell behaviors. Porcine ACL fibroblasts were obtained from explant culture and suspended in platelet poor plasma (PPP), 1× platelet-rich plasma (PRP), 3× PRP, 5× PRP, or phosphate buffered saline (PBS). The cell suspensions were cultured in a 3D collagen scaffold. Cellular metabolism (MTT assay), apoptosis (TUNEL assay), and gene expression for type I and type III collagen were measured. 1× PRP significantly outperformed 5× PRP in all parameters studied: Type I and III collagen gene expression, apoptosis prevention, and cell metabolism stimulation. ACL fibroblasts cultured with 1× PRP had the highest type I and type III collagen gene expression. 1× PRP and PPP groups had the highest cell metabolism and lowest apoptosis rates. Concentration of platelets had significant effects on the behavior of ACL fibroblasts; thus, it is an important parameter that should be specified in clinical or basic science studies.

  7. Platelets in the neonatal period: developmental differences in platelet production, function, and hemostasis and the potential impact of therapies.

    PubMed

    Sola-Visner, Martha

    2012-01-01

    Thrombocytopenia is a common problem among sick neonates admitted to the neonatal intensive care unit. Frequently, platelet transfusions are given to thrombocytopenic infants in an attempt to decrease the incidence or severity of hemorrhage, which is often intracranial. Whereas there is very limited evidence to guide platelet transfusion practices in this population, preterm infants in the first week of life (the highest risk period for bleeding) are nearly universally transfused at higher platelet counts than older infants or children. To a large extent, this practice has been influenced by the observation that neonatal platelets are hyporeactive in response to multiple agonists in vitro, although full-term infants exhibit normal to increased primary hemostasis. This apparently paradoxical finding is due to factors in the neonatal blood that enhance the platelet-vessel wall interaction and counteract the platelet hyporeactivity. Relatively few studies have evaluated the platelet function and primary hemostasis of preterm infants, the subset of neonates at highest risk of bleeding and those most frequently transfused. Current understanding of platelet production and function in preterm and full-term neonates, how these factors affect their response to thrombocytopenia and their primary hemostasis, and the implications of these developmental differences to transfusion medicine are reviewed herein.

  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 function tests in clinical cardiology: unfulfilled expectations.

    PubMed

    Gorog, Diana A; Fuster, Valentin

    2013-05-28

    This review is a critical evaluation of publications in the past decade on the usefulness of platelet function tests (PFTs) in clinical cardiology, in aiding diagnosis, predicting risk, and monitoring therapy. The ideal PFT should: 1) detect baseline platelet hyperreactivity; 2) allow individualization of antiplatelet medication; 3) predict thrombotic risk; and 4) predict bleeding risk. The practicalities of clinical cardiology demand rapid, accurate, and reliable tests that are simple to operate at the bedside and available 24 h a day, 7 days a week. Point-of-care PFTs most widely evaluated clinically include PFA-100 and VerifyNow. None of these tests can reliably detect platelet hyperreactivity and thus identify a prothrombotic state. Identification of antiplatelet nonresponsiveness or hyporesponsiveness is highly test specific, and does not allow individualization of therapy. The power of PFTs in predicting thrombotic events for a given individual is variable and often modest, and alteration of antithrombotic treatment on the basis of the results of PFTs has not been shown to alter clinical outcome. PFTs in current mainstream use cannot reliably assess bleeding risk. These tests have been in use for over a decade, but the hopes raised by PFTs in clinical practice remain unfulfilled. Although physiologically relevant measurement of platelet function now is more important than ever, a critical reappraisal of available techniques in light of clinical requirements is needed. The use of native blood, global stimulus instead of individual agonists, contribution of thrombin generation by activated platelets to the test results, and establishment of a PFT therapeutic range for each antiplatelet drug should be considered and is discussed.

  10. Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder.

    PubMed

    Cai, Huili; Mullier, François; Frotscher, Birgit; Briquel, Marie-Elisabeth; Toussaint, Marie; Massin, Frédéric; Lecompte, Thomas; Latger-Cannard, Véronique

    2016-04-01

    Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

  11. Could Microparticles Be the Universal Quality Indicator for Platelet Viability and Function?

    PubMed

    Maurer-Spurej, Elisabeth; Chipperfield, Kate

    2016-01-01

    High quality means good fitness for the intended use. Research activity regarding quality measures for platelet transfusions has focused on platelet storage and platelet storage lesion. Thus, platelet quality is judged from the manufacturer's point of view and regulated to ensure consistency and stability of the manufacturing process. Assuming that fresh product is always superior to aged product, maintaining in vitro characteristics should preserve high quality. However, despite the highest in vitro quality standards, platelets often fail in vivo. This suggests we may need different quality measures to predict platelet performance after transfusion. Adding to this complexity, platelets are used clinically for very different purposes: platelets need to circulate when given as prophylaxis to cancer patients and to stop bleeding when given to surgery or trauma patients. In addition, the emerging application of platelet-rich plasma injections exploits the immunological functions of platelets. Requirements for quality of platelets intended to prevent bleeding, stop bleeding, or promote wound healing are potentially very different. Can a single measurable characteristic describe platelet quality for all uses? Here we present microparticle measurement in platelet samples, and its potential to become the universal quality characteristic for platelet production, storage, viability, function, and compatibility.

  12. Could Microparticles Be the Universal Quality Indicator for Platelet Viability and Function?

    PubMed Central

    Chipperfield, Kate

    2016-01-01

    High quality means good fitness for the intended use. Research activity regarding quality measures for platelet transfusions has focused on platelet storage and platelet storage lesion. Thus, platelet quality is judged from the manufacturer's point of view and regulated to ensure consistency and stability of the manufacturing process. Assuming that fresh product is always superior to aged product, maintaining in vitro characteristics should preserve high quality. However, despite the highest in vitro quality standards, platelets often fail in vivo. This suggests we may need different quality measures to predict platelet performance after transfusion. Adding to this complexity, platelets are used clinically for very different purposes: platelets need to circulate when given as prophylaxis to cancer patients and to stop bleeding when given to surgery or trauma patients. In addition, the emerging application of platelet-rich plasma injections exploits the immunological functions of platelets. Requirements for quality of platelets intended to prevent bleeding, stop bleeding, or promote wound healing are potentially very different. Can a single measurable characteristic describe platelet quality for all uses? Here we present microparticle measurement in platelet samples, and its potential to become the universal quality characteristic for platelet production, storage, viability, function, and compatibility. PMID:28053805

  13. Evaluation of Dried Storage of Platelets for Transfusion: Physiologic Integrity and Hemostatic Functionality

    DTIC Science & Technology

    1994-06-17

    ONR Grant No. N00014-92-J-1244 EVALUATION OF DRIED STORAGE OF PLATELETS FOR TRANSFUSION: PHYSIOLOGIC INTEGRITY AND HEMOSTATIC FUNCTIONALITY. Principal...protection for commercialization of the manufacture and use of lyophilized platelets in transfusion medicine is still pending in U.S. Patent Court and... platelets for correction of bleeding times in thrombocytopenic rabbits. Three different preparations, representing the platelets from a total of six units

  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-induced thrombin generation by the calibrated automated thrombogram assay is increased in patients with essential thrombocythemia and polycythemia vera.

    PubMed

    Panova-Noeva, Marina; Marchetti, Marina; Spronk, Henri Maria; Russo, Laura; Diani, Erika; Finazzi, Guido; Finazzi, Good; Salmoiraghi, Silvia; Rambaldi, Alessandro; Rambaldi, Aueesandrd; Barbui, Tiziano; Barbui, Titiano; Ten Cate, Hugo; Ten Cate, Huao; Falanga, Anna

    2011-04-01

    The platelet contribution to the thrombophilic state of patients with myeloproliferative neoplasms (MPNs), i.e., essential thrombocythemia (ET) and polycythemia vera (PV), remains uncertain. In this study we aimed to characterize the thrombin generation (TG) potential expressed by platelets from these subjects, compare it to normal platelets, and identify what factors might be responsible for platelet TG. In a group of 140 MPN patients (80 ET and 60 PV) and 72 healthy subjects, we measured the global procoagulant potential of platelet-rich plasma (PRP) utilizing the TG assay by the calibrated automated thrombogram (CAT). To characterize the procoagulant contribution of platelets in PRP, the TG of both isolated platelets and platelet-poor plasma was measured, and the platelet surface expression of TF was determined. Finally, the activation status of platelets was assessed by the levels of P-selectin expressed on platelet surface. MPN patients had significantly increased PRP and isolated platelet TG potential compared to controls. This was associated to the occurrence of platelet activation. Patients carriers of the JAK2V617F mutation showed the highest values of TG and platelet surface TF and P-selectin. Platelet TG potential was significantly lower in hydroxyurea(HU) compared to non-HU-treated patients and was lowest in HU-treated JAK2V617F carriers. In subjects not receiving HU, platelet TG significantly increased by JAK2V617F allele burden increment (P < 0.05).This study demonstrates a platelet-dependent form of hypercoagulability in MPN patients, particularly in those carriers of the JAK2V617F mutation. The cytoreductive therapy with HU significantly affects this prothrombotic phenotype.

  16. Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function.

    PubMed

    Lu, H; Menashi, S; Garcia, I; Cramer, E M; Li, H; Tenza, D; De Romeuf, C; Soria, J; Soria, C

    1993-09-01

    Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01-0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with < 0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min, indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31 +/- 0.90 for resting platelets, down-regulated to 0.84 +/- 0.13 (P < 0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63 +/- 2.21 (P > 0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.

  17. Migration distance-based platelet function analysis in a microfluidic system.

    PubMed

    Song, Suk-Heung; Lim, Chae-Seung; Shin, Sehyun

    2013-01-01

    Aggregation and adhesion of platelets to the vascular wall are shear-dependent processes that play critical roles in hemostasis and thrombosis at vascular injury sites. In this study, we designed a simple and rapid assay of platelet aggregation and adhesion in a microfluidic system. A shearing mechanism using a rotating stirrer provided adjustable shear rate and shearing time and induced platelet activation. When sheared blood was driven through the microchannel under vacuum pressure, shear-activated platelets adhered to a collagen-coated surface, causing blood flow to significantly slow and eventually stop. To measure platelet adhesion and aggregation, the migration distance (MD) of blood through the microchannel was monitored. As the microstirrer speed increased, MD initially decreased exponentially but then increased beyond a critical rpm. For platelet-excluded blood samples, there were no changes in MD with increasing stirrer speed. These findings imply that the stirrer provided sufficiently high shear to activate platelets and that blood MD is a potentially valuable index for measuring the shear-dependence of platelet activation. Our microfluidic system is quick and simple, while providing a precise assay to measure the effects of shear on platelet aggregation and adhesion.

  18. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.

    PubMed

    Rowley, Jesse W; Chappaz, Stéphane; Corduan, Aurélie; Chong, Mark M W; Campbell, Robert; Khoury, Amanda; Manne, Bhanu Kanth; Wurtzel, Jeremy G T; Michael, James V; Goldfinger, Lawrence E; Mumaw, Michele M; Nieman, Marvin T; Kile, Benjamin T; Provost, Patrick; Weyrich, Andrew S

    2016-04-07

    Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband β3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband β3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.

  19. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets

    PubMed Central

    Chappaz, Stéphane; Corduan, Aurélie; Chong, Mark M. W.; Campbell, Robert; Khoury, Amanda; Manne, Bhanu Kanth; Wurtzel, Jeremy G. T.; Michael, James V.; Goldfinger, Lawrence E.; Mumaw, Michele M.; Nieman, Marvin T.; Kile, Benjamin T.; Provost, Patrick; Weyrich, Andrew S.

    2016-01-01

    Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIb and β3 mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3′ untranslated region luciferase reporter assays confirmed that the translation of both αIIb and β3 mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. Combined Pf4-cre–mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets. PMID:26773046

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

    PubMed

    Kaneez, Fatima Shad; Saeed, Sheikh Arshad

    2009-08-01

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

  1. Ibrutinib treatment affects collagen and von Willebrand factor-dependent platelet functions.

    PubMed

    Levade, Marie; David, Elodie; Garcia, Cédric; Laurent, Pierre-Alexandre; Cadot, Sarah; Michallet, Anne-Sophie; Bordet, Jean-Claude; Tam, Constantine; Sié, Pierre; Ysebaert, Loïc; Payrastre, Bernard

    2014-12-18

    The oral Bruton's tyrosine kinase inhibitor, ibrutinib, has recently demonstrated high efficiency in patients with relapsed B-cell malignancies. Occurrence of bleeding events has been reported in a subgroup of ibrutinib-treated patients. We demonstrate that ibrutinib selectively inhibits platelet signaling and functions downstream of the collagen receptor glycoprotein VI and strongly affects firm platelet adhesion on von Willebrand factor (VWF) under arterial flow. A longitudinal study of 14 patients indicated a correlation between occurrence of bleeding events and decreased platelet aggregation in response to collagen in platelet-rich plasma and firm adhesion on VWF under arterial flow. The addition of 50% untreated platelets was sufficient to efficiently reverse the effects of ibrutinib, and platelet functions recovered after treatment interruption as physiological platelet renewal occurred. These data have important clinical implications and provide a basis for hemostasis management during ibrutinib treatment.

  2. 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

  3. The use of regression analysis in determining reference intervals for low hematocrit and thrombocyte count in multiple electrode aggregometry and platelet function analyzer 100 testing of platelet function.

    PubMed

    Kuiper, Gerhardus J A J M; Houben, Rik; Wetzels, Rick J H; Verhezen, Paul W M; van Oerle, Rene; Ten Cate, Hugo; Henskens, Yvonne M C; Lancé, Marcus D

    2017-01-09

    Low platelet counts and hematocrit levels hinder whole blood point-of-care testing of platelet function. Thus far, no reference ranges for MEA (multiple electrode aggregometry) and PFA-100 (platelet function analyzer 100) devices exist for low ranges. Through dilution methods of volunteer whole blood, platelet function at low ranges of platelet count and hematocrit levels was assessed on MEA for four agonists and for PFA-100 in two cartridges. Using (multiple) regression analysis, 95% reference intervals were computed for these low ranges. Low platelet counts affected MEA in a positive correlation (all agonists showed r(2) ≥ 0.75) and PFA-100 in an inverse correlation (closure times were prolonged with lower platelet counts). Lowered hematocrit did not affect MEA testing, except for arachidonic acid activation (ASPI), which showed a weak positive correlation (r(2) = 0.14). Closure time on PFA-100 testing was inversely correlated with hematocrit for both cartridges. Regression analysis revealed different 95% reference intervals in comparison with originally established intervals for both MEA and PFA-100 in low platelet or hematocrit conditions. Multiple regression analysis of ASPI and both tests on the PFA-100 for combined low platelet and hematocrit conditions revealed that only PFA-100 testing should be adjusted for both thrombocytopenia and anemia. 95% reference intervals were calculated using multiple regression analysis. However, coefficients of determination of PFA-100 were poor, and some variance remained unexplained. Thus, in this pilot study using (multiple) regression analysis, we could establish reference intervals of platelet function in anemia and thrombocytopenia conditions on PFA-100 and in thrombocytopenia conditions on MEA.

  4. THE EFFECT OF ACETYLSALICYLIC ACID ON PLATELET FUNCTION

    PubMed Central

    Evans, Geoffrey; Packham, Marian A.; Nishizawa, Edward E.; Mustard, James F.; Murphy, Edmund A.

    1968-01-01

    Acetylsalicylic acid (ASA, aspirin) and sodium salicylate inhibit platelet aggregation induced by collagen, antigen-antibody complexes, gamma globulin-coated particles or thrombin. These compounds suppress the release of platelet constituents, such as adenosine diphosphate (ADP) and serotonin, induced by such stimuli. Since ASA and sodium salicylate do not inhibit ADP-induced platelet aggregation, it appears that their effect on the action of the other stimuli is due to a decrease in the amount of ADP released. The administration of ASA to rabbits (in doses which inhibited collagen-induced platelet aggregation) impaired hemostasis, prolonged platelet survival, and diminished the amount of deposit formed in an extracorporeal shunt. PMID:4176225

  5. Plasma protein regulation of platelet function and metabolism.

    PubMed

    Hansen, M S; Bang, N U

    1979-04-02

    This reviews summarizes our evidence suggesting that the plasma protein enviroment influences platelet aggregation potential and metabolic activity. Cationic proteins are capable of restoring the aggreation potential of washed human platelets. The aggregation restoring effect of gamma globulin is inhibited by more anionic proteins in subfractions of Cohn fraction IV and fractions V and VI. Artificial enhancement of the net negative charge of plasma proteins through acylation produces derivatives capable of inhibiting platelet rich plasma. The oxygen consumption of washed human platelets is lower than in platelet rich plasma while the lactate production is identical. Autologus plasma, albumin or IgG immunoglobulin restores the oxygen consumption of washed platelets to values comparable to those obtained for platelet rich plasma, while the lactate production is unaffected. Fibrinogen on IgA myeloma protein increases the lactate production, but not the oxygen consumption. Cyclic AMP levels are considerably lower in washed platelets than in platelet rich plasma. Gamma globulin and albumin causes a futher decrease, which is progressive with time. Fibrinogen causes no change in platelet cyclic AMP content. It is suggested that these observations may in part be explained by the equilibriun between anionic and cationic proteins in the platelet microenvironment. This hypothesis appears applicable in certain situations.

  6. 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.

  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. [Morphology, ultrastructure and function of glycosylation-modified chilled blood platelets].

    PubMed

    Guo, Yong; Han, Ying; Quan, Guo-Bo; Liu, Min-Xia; Liu, An

    2008-04-01

    The glycosylation of platelets may prolong their life-span when being transfused after preservation under 4 degrees C, therefore this study was aimed to investigate the effect of glycosylation on morphology, ultrastructure, function and membrane glycoprotein of platelets. The experiments were divided into 3 groups: group preserved in room temperature (RT group), group preserved in 4 degrees C (4T group) and group UDP-Gal glycosylated and preserved in 4 degrees C (U+4T group). The binding rate of RCA I lectin and expression of platelet surface markers CD62P, CD42b were determined by flow cytometry. Morphology and ultrastructure of platelets were observed by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Platelets aggregation was detected by aggregometer. The results showed that the binding rate of RCAI in U+4T group significantly higher than that in RT group (p<0.01), no obvious changes was found in ultrastructure of glycosylated platelets, as compared with fresh platelets. Some morphologic changes, such as pseudopodium could be observed in 4T group. The aggregation rate of platelets in U+4T group reached to 50% of RT group. The expression levels of CD42b and CD62P, and the binding rate of annexin V in U+4T group were not significantly different from that in RT group. It is concluded that UDP-Gal can effectively cause galactosylation of platelets, and the platelets modified with UDP-Gal remain normal morphology, ultrastructure and function.

  9. Platelets possess functional TGF-beta receptors and Smad2 protein.

    PubMed

    Lev, P R; Salim, J P; Marta, R F; Osorio, M J Mela; Goette, N P; Molinas, F C

    2007-02-01

    TGF-beta1 plays a main role in tissue repair by regulating extracellular matrix production and tissue granulation. Platelets are one of the main sources of this cytokine in the circulation. The aim of this study was to evaluate the presence of the TGF-beta receptors on platelets, the effect of TGF-beta1 on platelet aggregation and the underlying intracellular mechanisms. TGF-beta receptors on platelets were studied by flow cytometry and their mRNA by PCR. Platelet aggregation was assessed by turbidimetric methods and intracellular pathways by Western blot. TGF-beta receptor type II and mRNA codifying for TbetaRI and TbetaRII were found in platelets. We demonstrated that TGF-beta1 did not trigger platelet aggregation by itself but had a modulating effect on ADP-induced platelet aggregation. Either inhibition or increase in platelet aggregation, depending on the exposure time to TGF-beta1 and the ADP concentration used, were shown. We found that platelets possess Smad2 protein and that its phosphorylation state is increased after exposure to TGF-beta1. Besides, TGF-beta1 modified the pattern of ADP-induced tyrosine phosphorylation. Increased phosphorylation levels of 64-, 80- and 125-kDa proteins during short time incubation with TGF-beta1 and increased phosphorylation of 64- and 125-kDa proteins after longer incubation were observed. The modulating effect of TGF-beta1 on platelet aggregation could play a role during pathological states in which circulating TGF-beta1 levels are increased and intravascular platelet activation is present, such as myeloproliferative disorders. In vascular injury, in which platelet activation followed by granule release generates high local ADP concentrations, it could function as a physiological mechanism of platelet activation control.

  10. Beta-lactam antibiotic-mediated changes in platelet reactivity and vascular endothelial functions.

    PubMed

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

    2001-05-01

    To evaluate vascular and platelet compatibility of intravenous administration of beta-lactam antibiotics, we assessed the effects of therapeutic concentrations of ceftriaxone, aztreonam, and ceftazidime on platelet reactivity to different agonists (sodium arachidonate, collagen and adenosine diphosphate) and on selected vascular endothelial functions (adenosine diphosphatase activity, prostacyclin production and t-PA release). Ceftriaxone and, to a lesser degree, aztreonam, enhanced platelet reactivity, evaluated as onset of platelet aggregating response, and increased thromboxane production to subthreshold concentrations of arachidonate. There was no modification in platelet reactivity after ceftazidime treatment. Ceftriaxone and ceftazidime, but not aztreonam, inhibited endothelial adenosine diphosphatase activity. Prostacyclin production and t-PA release were inhibited only by ceftriaxone at high concentrations. While it is difficult to establish which marker (platelet or endothelial functions) has more clinical reference in human vascular compatibility, it seems feasible to consider aztreonam the most compatible of the beta-lactams studied.

  11. NF-E2 p45 is important for establishing normal function of platelets.

    PubMed

    Fujita, Rie; Takayama-Tsujimoto, Mariko; Satoh, Hironori; Gutiérrez, Laura; Aburatani, Hiroyuki; Fujii, Satoshi; Sarai, Akinori; Bresnick, Emery H; Yamamoto, Masayuki; Motohashi, Hozumi

    2013-07-01

    NF-E2 is a heterodimeric transcription factor consisting of p45 and small Maf subunits. Since p45(-/-) mice display severe thrombocytopenia, p45 is recognized as a critical regulator of platelet production from megakaryocytes. To identify direct p45 target genes in megakaryocytes, we used chromatin immunoprecipitation (ChIP) sequencing to analyze the genome-wide chromatin occupancy of p45 in primary megakaryocytes. p45 target gene candidates obtained from the analysis are implicated in the production and function of platelets. Two of these genes, Selp and Myl9, were verified as direct p45 targets through multiple approaches. Since P-selectin, encoded by Selp, plays a critical role in platelet function during thrombogenesis, we tested whether p45 determines the intrinsic reactivity and potency of platelets generated from megakaryocytes. Mice expressing a hypomorphic p45 mutant instead of wild-type p45 in megakaryocytes (p45(-/-):ΔNTD-Tg mice) displayed platelet hypofunction accompanied by mild thrombocytopenia. Furthermore, lung metastasis of melanoma cells, which requires platelet activation, was repressed in p45(-/-):ΔNTD-Tg mice compared to control mice, validating the impaired function of platelets produced from p45(-/-):ΔNTD-Tg megakaryocytes. By activating genes in megakaryocytes that mediate platelet production and function, p45 determines the quantity and quality of platelets.

  12. NF-E2 p45 Is Important for Establishing Normal Function of Platelets

    PubMed Central

    Fujita, Rie; Takayama-Tsujimoto, Mariko; Satoh, Hironori; Gutiérrez, Laura; Aburatani, Hiroyuki; Fujii, Satoshi; Sarai, Akinori; Bresnick, Emery H.

    2013-01-01

    NF-E2 is a heterodimeric transcription factor consisting of p45 and small Maf subunits. Since p45−/− mice display severe thrombocytopenia, p45 is recognized as a critical regulator of platelet production from megakaryocytes. To identify direct p45 target genes in megakaryocytes, we used chromatin immunoprecipitation (ChIP) sequencing to analyze the genome-wide chromatin occupancy of p45 in primary megakaryocytes. p45 target gene candidates obtained from the analysis are implicated in the production and function of platelets. Two of these genes, Selp and Myl9, were verified as direct p45 targets through multiple approaches. Since P-selectin, encoded by Selp, plays a critical role in platelet function during thrombogenesis, we tested whether p45 determines the intrinsic reactivity and potency of platelets generated from megakaryocytes. Mice expressing a hypomorphic p45 mutant instead of wild-type p45 in megakaryocytes (p45−/−:ΔNTD-Tg mice) displayed platelet hypofunction accompanied by mild thrombocytopenia. Furthermore, lung metastasis of melanoma cells, which requires platelet activation, was repressed in p45−/−:ΔNTD-Tg mice compared to control mice, validating the impaired function of platelets produced from p45−/−:ΔNTD-Tg megakaryocytes. By activating genes in megakaryocytes that mediate platelet production and function, p45 determines the quantity and quality of platelets. PMID:23648484

  13. Effect of Fibrinogen on Platelet Reactivity Measured by the VerifyNow P2Y12 Assay.

    PubMed

    Dobrovolsky, A B; Laguta, P S; Guskova, E V; Yarovaya, E B; Titaeva, E V; Storozhilova, A N; Panchenko, E P

    2016-05-01

    The VerifyNow assay is based upon the ability of activated platelets to cross-link beads coated with fibrinogen. However, fibrinogen is an abundant protein of blood, and therefore it may affect test results by competing with fibrinogen of beads for binding to platelets. To test this assumption, we assessed the influence of artificial alteration of fibrinogen level in blood samples obtained from donors (n = 9) and patients on clopidogrel therapy (n = 8) on the results of the VerifyNow P2Y12 assay. Fibrinogen level was altered by adding to blood samples 1/10 volume of fibrinogen solution (10.56 g/liter) or corresponding buffer. Relative to baseline, addition of buffer significantly increased platelet reactivity, whereas addition of fibrinogen decreased it. Analysis of the relationship between change in platelet reactivity values (dBase and dPRU) and change in fibrinogen concentration (dFg) revealed strong negative correlations: dBase = -63.3 × dFg - 27.1 (r = -0.924, p < 0.0005) and dPRU = -54.4 × dFg - 21.8 (r = -0.764, p < 0.0005). Thus, the results of our experiments suggest that: (i) blood fibrinogen strongly influences results of the VerifyNow P2Y12 assay, and (ii) correcting for fibrinogen effect may be needed to improve the accuracy of the test in the measuring of antiplatelet effect of clopidogrel therapy.

  14. The sticky platelet syndrome.

    PubMed

    Moncada, Benjamín; Ruíz-Arguelles, Guillermo J; Castillo-Martínez, Claudio

    2013-07-01

    The sticky platelets syndrome (SPS) is a procoagulant condition based on either arterial, venous, or capillary thrombi caused by hyperesponsive and hyperaggregable platelets. This is a frequent disease, which often remains clinically inapparent, until stressful events or combination with other factors increase the risk of developing SPS. The condition is due to a congenital platelet defect with autosomal dominant characteristics, leading to the increased platelet aggregability when they are challenged with epinephrine and adenosine diphosphate. Nowadays classification of this disorder is based on platelet reactivity to both ADP and epinephrine (SPS type 1), epinephrine alone (SPS type 2), and ADP alone (SPS type 3). The diagnoses of the syndrome depend on the functional aggregometer assay. This condition should be taken into account whenever a patient with thrombophilia is considered.

  15. Platelet function and activation in Cavalier King Charles Spaniels with subclinical chronic valvular heart disease.

    PubMed

    Tong, Linda J; Hosgood, Giselle L; French, Anne T; Irwin, Peter J; Shiel, Robert E

    2016-08-01

    OBJECTIVE To assess platelet closure time (CT), mean platelet component (MPC) concentration, and platelet component distribution width (PCDW) in dogs with subclinical chronic valvular heart disease. ANIMALS 89 Cavalier King Charles Spaniels (CKCSs) and 39 control dogs (not CKCSs). PROCEDURES Platelet count, MPC concentration, PCDW, and Hct were measured by use of a hematology analyzer, and CT was measured by use of a platelet function analyzer. Murmur grade and echocardiographic variables (mitral valve regurgitant jet size relative to left atrial area, left atrial-to-aortic diameter ratio, and left ventricular internal dimensions) were recorded. Associations between explanatory variables (sex, age, murmur grade, echocardiographic variables, platelet count, and Hct) and outcomes (CT, MPC concentration, and PCDW) were examined by use of multivariate regression models. RESULTS A model with 5 variables best explained variation in CT (R(2), 0.74), with > 60% of the variance of CT explained by mitral valve regurgitant jet size. The model of best fit to explain variation in MPC concentration included only platelet count (R(2), 0.24). The model of best fit to explain variation in PCDW included platelet count and sex (R(2), 0.25). CONCLUSIONS AND CLINICAL RELEVANCE In this study, a significant effect of mitral valve regurgitant jet size on CT was consistent with platelet dysfunction. However, platelet activation, as assessed on the basis of the MPC concentration and PCDW, was not a feature of subclinical chronic valvular heart disease in CKCSs.

  16. Inhibitory effects of cardiotonic pills on platelet function in dogs fed a high-fat diet.

    PubMed

    Zhang, Lei; Zheng, Jun; Li, Hui-Min; Meng, Yong-Xia

    2006-06-01

    Insulin resistance and the consequent metabolic disorders are associated with a state of platelet hyperactivity. Oxidative stress is responsible for the persistent platelet activation. We sought to study the inhibitory effect of cardiotonic pills, an oral herbal component, on platelet function in a dog model with insulin resistance induced by high-fat feeding. We fed 18 dogs with a high-fat diet and six dogs with normal chow as control for 6 months. Then, six dogs were fed with a high-fat diet and received additional aspirin (250 mg/day), and another six dogs received additional cardiotonic pills (1,000 mg/day) for 4 months. Time-course changes in metabolic parameters and platelet function were detected. After high-fat feeding for 6 months, 18 dogs developed a series of metabolic disorders including obesity, dyslipidemia, oxidative stress and insulin resistance. In addition, a platelet hyperactivity state, characterized by increased agonist (arachidonic acid, ADP and collagen) induced platelet aggregation, platelet expression of adhesion molecules (P-selectin and GP IIb/IIIa), and platelet intracellular calcium concentration, was indicated. Cardiotonic pills showed a significant antioxidative activity by presenting an increase in plasma superoxide dismutase and decrease in erythrocyte glutathione, as well as a lipid-lowering effect (decrease in both plasma cholesterol and triglyceride). Either aspirin or cardiotonic pills could significantly reverse the platelet hypersensitivity and hyperfunction. Compared with aspirin, cardiotonic pills showed a more exaggerated inhibitory effect on platelet function (a significantly decreased collagen-stimulated platelet aggregation, and expression of adhesion molecules). In conclusion, cardiotonic pills inhibited platelet hyperfunction in dogs with insulin resistance. This inhibitory effect may mainly be explained by antioxidative activity and metabolic control.

  17. Intrinsic platelet reactivity before start with clopidogrel as predictor for on-clopidogrel platelet function and long-term clinical outcome.

    PubMed

    Hochholzer, Willibald; Valina, Christian M; Bömicke, Timo; Amann, Michael; Stratz, Christian; Nührenberg, Thomas; Trenk, Dietmar; Neumann, Franz-Josef

    2015-07-01

    High on-clopidogrel platelet reactivity is associated with worse clinical outcome. Previous data suggest that intrinsic platelet reactivity before initiation of clopidogrel contributes significantly to on-clopidogrel platelet reactivity. It is unknown whether intrinsic reactivity can sufficiently predict on-clopidogrel reactivity and therefore identify patients with insufficient response to clopidogrel before initiation of treatment and at risk for worse clinical outcome. This analysis included 765 consecutive patients undergoing elective coronary stent implantation. Platelet reactivity was assessed by light transmission aggregometry (5 µM ADP) before administration of clopidogrel 600mg and after intake of first maintenance dose of clopidogrel on day 1 following coronary stenting. Patients were followed for up to seven years. The combined primary endpoint was death of any cause or non-fatal myocardial infarction. Intrinsic and on-clopidogrel platelet reactivity were significant correlated (r=0.31; p < 0.001). Among all tested clinical and genetic factors including the cytochrome P450 2C19*2 polymorphism, intrinsic platelet reactivity was the strongest predictor for on-clopidogrel platelet reactivity. However, intrinsic platelet reactivity could only explain 8 % of variability of on-clopidogrel platelet function. Only on-treatment platelet reactivity was predictive for long-term clinical outcome (HR 1.47, 95 % CI 1.05-2.05; p = 0.02) whereas intrinsic platelet reactivity was not (HR 1.03, 95 % CI 0.74-1.43; p = 0.86). In conclusion, intrinsic platelet reactivity before initiation of clopidogrel is the strongest predictor of early on-clopidogrel platelet reactivity but can only explain a minor proportion of its variability and is not significantly associated with clinical outcome. Thus, baseline testing cannot substitute on-clopidogrel platelet function testing.

  18. Unravelling the different functions of protein kinase C isoforms in platelets.

    PubMed

    Heemskerk, Johan W M; Harper, Matthew T; Cosemans, Judith M E M; Poole, Alastair W

    2011-06-23

    Platelets tightly regulate haemostasis and arterial thrombosis. Protein kinase C (PKC) is involved in most platelet responses implicated in thrombus formation. Recent pharmacological and mouse gene knockout approaches show that the conventional PKC isoforms and the novel PKC isoforms contribute in distinct ways to these platelet responses. We hypothesize that, in platelets and other cells, the characteristic functions of PKC isoforms are established through unique activation mechanisms and unique interacting protein partners, which result in isoform-specific patterns of substrate phosphorylation. For identifying the substrate proteins in a living cell, new methodology is available and discussed.

  19. Evaluation of store lesion in platelet obtained by apheresis compared to platelet derived from whole blood and its impact on the in vitro functionality.

    PubMed

    Quintero, M; Núñez, M; Mellado, S; Maldonado, M; Wehinger, S

    2015-12-01

    Platelet units for transfusion purposes are obtained manually from whole blood or by apheresis, in an automated process. In both methods, platelets during storage present a characteristics grouped under the name "storage lesion" that are associated with adverse effects on platelet units. Oxidative stress has been claimed to be one of major causes, leading to activation and apoptosis processes affecting their post transfusion functionality. In this work, we observed an association between apheresis and a reduced presence of oxidative stress and better results in functional markers in stored platelets, compared to manually obtained platelets. Then, apheresis which would ensure a greater number of functional platelets during the 5 days of storage, compared to concentrates obtained from whole blood.

  20. Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling.

    PubMed

    Buitrago, Lorena; Langdon, Wallace Y; Sanjay, Archana; Kunapuli, Satya P

    2011-11-17

    c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-Cbl(YF/YF) knock-in mice. c-Cbl KO and c-Cbl(YF/YF) platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl(YF/YF) platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.

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

    PubMed Central

    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

  2. Hemostatic Function of Apheresis Platelets Stored at 4 deg C and 22 deg C

    DTIC Science & Technology

    2014-05-01

    function (5). Cold storage at 4-C could prolong shelf life by diminishing the risk of bacterial sepsis , decreasing platelet metabolism, and...glucose and lactate accumulation that occurs from continuous metabolic activity (37, 38). These markers represent different molecular events of...These data suggest that distinct mechanisms of activation are triggered at each storage temperature tested. While platelet activation at RT is

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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

  8. Posttranslational Modification Assays on Functional Protein Microarrays.

    PubMed

    Neiswinger, Johnathan; Uzoma, Ijeoma; Cox, Eric; Rho, HeeSool; Jeong, Jun Seop; Zhu, Heng

    2016-10-03

    Protein microarray technology provides a straightforward yet powerful strategy for identifying substrates of posttranslational modifications (PTMs) and studying the specificity of the enzymes that catalyze these reactions. Protein microarray assays can be designed for individual enzymes or a mixture to establish connections between enzymes and substrates. Assays for four well-known PTMs-phosphorylation, acetylation, ubiquitylation, and SUMOylation-have been developed and are described here for use on functional protein microarrays. Phosphorylation and acetylation require a single enzyme and are easily adapted for use on an array. The ubiquitylation and SUMOylation cascades are very similar, and the combination of the E1, E2, and E3 enzymes plus ubiquitin or SUMO protein and ATP is sufficient for in vitro modification of many substrates.

  9. 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.

  10. Measurement of platelet aggregation functions using whole blood migration ratio in a microfluidic chip.

    PubMed

    Seo, Hong Seog; Choi, Sung Hyuk; Han, Miran; Kim, Kyeong Ah; Cho, Chi Hyun; An, Seong Soo A; Lim, Chae Seung; Shin, Sehyun

    2016-01-01

    Platelets play a major role in maintaining endothelial integrity and hemostasis. Of the various soluble agonists, ADP is an important in vivo stimulus for inducing platelet aggregation. In this study, a simple, rapid, and affordable method was designed for testing bleeding time (BT) and platelet aggregation with a two-channel microfluidic chip. Whole blood migration ratio (MR) from a microchip system was evaluated in comparison to the closure time (CT) from PFA-100 assays (Siemens, Germany) and CD62P expression on platelets. To induce platelet aggregation, a combination of collagen (1.84 mg/ml) and ADP (37.5 mg/ml) were used as agonists. After adding the agonists to samples, whole blood MR from the microchip system was measured. The outcome of the assessment depended on reaction time and agonist concentration. MR of whole blood from the microchip system was significantly correlated with CT from PFA-100 (r = 0.61, p <  0.05, n = 60). In addition, MR was negatively correlated with CD62P expression (r =-0.95, p <  0.05, n = 60). These results suggest that the measurement of MR using agonists is an easy, simple and efficient method for monitoring platelet aggregation in normal and ADP-receptors defective samples, along with the BT test. Thus, usage of the current microfluidic method could expand to diverse applications, including efficacy assessments in platelet therapy.

  11. 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

  12. CEACAM1 regulates integrin αIIbβ3-mediated functions in platelets.

    PubMed

    Yip, Jana; Alshahrani, Musaed; Beauchemin, Nicole; Jackson, Denise E

    2016-01-01

    Previous studies have implicated that the Ig-ITIM superfamily member, CEACAM1 may regulate integrin function. While CEACAM1 has been demonstrated to play a role as an inhibitory co-receptor of ITAM-associated GPVI/FcR γ-chain signaling pathways in platelets, its physiologic role in integrin αIIbβ3-mediated platelet function is unclear. In this study, we investigate the functional importance of Ceacam1 in murine platelets. We show that CEACAM1 is constitutively associated with integrin αIIbβ3 in resting human and mouse platelets as demonstrated by co-immunoprecipitation studies. Using Ceacam1-deficient mice, we show that they have prolonged tail bleeding times and volume of blood lost that is corrected by reconstitution with platelet Ceacam1. Ceacam1(-/-) platelets have moderate integrin αIIbβ3 mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and failure to retract fibrin clots in vitro. This functional integrin αIIbβ3 defect could not be attributed to altered integrin αIIbβ3 expression. Ceacam1(-/-) platelets displayed normal "inside-out" signaling properties as demonstrated by normal agonist-induced binding of soluble (fluorescein isothiocyanate) FITC-fibrinogen, JON/A antibody binding, and increases in cytosolic free calcium levels. This study provides direct evidence that Ceacam1 is essential for normal integrin αIIbβ3-mediated platelet function and that disruption of mouse Ceacam1 induced moderate integrin αIIbβ3-mediated functional defects.

  13. Normal platelet function in platelet concentrates requires non-platelet cells: a comparative in vitro evaluation of leucocyte-rich (type 1a) and leucocyte-poor (type 3b) platelet concentrates

    PubMed Central

    Parrish, William R; Roides, Breana; Hwang, Julia; Mafilios, Michael; Story, Brooks; Bhattacharyya, Samir

    2016-01-01

    Background Therapeutic success of platelet-rich plasma (PRP) may vary based on the composition and preparation method. The objective of this study was to evaluate the cellular components of platelet concentrates produced by a leucocyte-rich (LR-PRP) and a leucocyte-poor PRP systems (LP-PRP). Methods Parameters evaluated included platelet recovery, platelet concentration, red blood cell (RBC) and white blood cell (WBC) composition, platelet growth factor release and stimulation of human tendon cell proliferation in vitro. Results Platelet recoveries were 52% for LP-PRP and 89% for LR-PRP. LR-PRP demonstrated greater reproducibility with a 4.2% coefficient of variation (CV) compared with 19.4% for LP-PRP (p<0.001). LR-PRP demonstrated a greater increase in platelet concentration (7.9-fold) than LP-PRP (2.2-fold; p<0.001). LP-PRP showed 5.0-fold reductions in WBCs, while LR-PRP showed a 4.0-fold increase (p<0.001). LP-PRP reduced RBCs to a haematocrit of 0.25, while LR-PRP reduced haematocrit to 11.8. LP-PRP did not coagulate robustly on reactivation with CaCl2, and released significantly lower levels of epidermal growth factor (EGF) and transforming growth factor β1 (TGF-β1) than whole blood (p<0.03). LP-PRP also did not stimulate tendon cell proliferation greater than whole blood. In contrast, LR-PRP showed increases in each growth factor on activation with CaCl2 (p<0.01) and stimulated greater proliferation (p<0.05) compared with whole blood. Forced activation of LP-PRP with exogenous thrombin rescued the coagulation deficiency and induced greater growth factor release than comparable whole blood (p<0.03). Conclusions These data suggest that non-platelet cellular components in platelet concentrates are important for proper platelet function, including thrombin generation, growth factor release and clot retraction. PMID:27900155

  14. 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

  15. Expression of the PlA2 allele of glycoprotein IIIa and its impact on platelet function

    PubMed Central

    Ferro, Albert; Warner, Timothy D

    2015-01-01

    Background The platelet fibrinogen receptor represents the final common pathway of platelet activation, and is formed from two glycoprotein (GP) subunits (GPIIb/IIIa). Carriage of the mutant PlA2 allele of GPIIIa has been shown to confer an increased risk of cardiovascular events, but published studies have disagreed as to the mechanism for this association. Objectives To assess whether carriage of the PlA2 allele conforms to Mendelian patterns of expression and to identify whether carriage of the mutant allele modulates platelet function. Methods Expression of the PlA2 allele was assessed in both healthy subjects (n = 25) and patients with known coronary artery disease (n = 90) through the development and validation of a liquid chromatography, tandem mass spectrometry (LC-MS/MS) assay. Platelet function was assessed in the patient cohort in response to multiple agonists, and these data were analysed in the context of the proteomic data. Results Expression of the wild-type PlA1 allele and mutant PlA2 alleles was readily quantifiable and conformed to Mendelian patterns in both healthy and patient cohorts. Patients who were homozygous for the mutant PlA2 allele had an increased aggregatory response to adenosine diphosphate, collagen, adrenaline, ristocetin, thrombin receptor-activating peptide 6 and U46619, when assessed using agonist-concentration response curves. Conclusions These findings support the hypothesis that carriage of the mutant PlA2 allele mediates an increased risk of cardiovascular events through the modulation of platelet reactivity. PMID:26858830

  16. Bleeding tendency and platelet function during treatment with romiplostim in children with severe immune thrombocytopenic purpura.

    PubMed

    Suntsova, Elena V; Demina, Irina M; Ignatova, Anastasia A; Ershov, Nikolay M; Trubina, Natalia M; Dobrynina, Juliya; Serkova, Irina V; Supik, Zhanna S; Orekhova, Ekaterina V; Hachatryan, Lili A; Kotskaya, Natalia N; Pshonkin, Aleksey V; Maschan, Aleksey A; Novichkova, Galina A; Panteleev, Mikhail A

    2017-03-07

    It has been suggested that platelet function in chronic immune thrombocytopenic purpura (ITP) may be abnormal. Thrombopoietin mimetics used for treatment can affect it, but the data remain limited. We investigated platelet function of 20 children diagnosed with severe ITP (aged 1-16 years, 12 females and eight males). Platelet functional activity in whole blood was characterized by flow cytometry before and after stimulation with SFLLRN plus collagen-related peptide. Levels of CD42b, PAC1, and CD62P, but not CD61 or annexin V, were significantly increased (P < 0.05) in resting platelets of patients before treatment compared with healthy donors. On average, PAC1 and CD62P in patients after activation were also significantly elevated, although some patients failed to activate integrins. Romiplostim (1-15 μg/kg/week s.c.) was prescribed to seven patients, with clinical improvement in six. Interestingly, one patient had clinical improvement without platelet count increase. Eltrombopag (25-75 mg/day p.o.) was given to four patients, with positive response in one. Others switched to romiplostim, with one stable positive response, one unstable positive response, and one non-responding. Platelet quality improved with romiplostim treatment, and their parameters approached the normal values. Our results suggest that platelets in children with severe ITP are pre-activated and abnormal, but improve with treatment.

  17. 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.

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

    PubMed

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

    2010-09-21

    IIbβ3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function. A critical function of platelets is to adhere to regions of damage on blood vessel walls; in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix interactions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

  19. Hyperthermia inhibits platelet haemostatic functions and selectively regulates the release of alpha-granule proteins

    PubMed Central

    Etulain, J; Lapponi, MJ; Patrucchi, SJ; Romaniuk, MA; Benzadón, R; Klement, GL; Negrotto, S; Schattner, M

    2011-01-01

    Summary Background Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompany these processes, but very little is known about the changes in platelet function that occur at different temperatures. Objectives To explore the effect of higher temperatures on platelet physiology. Methods Platelet responses including adhesion, spreading (fluorescence microscopy), αIIbbeta;3 activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A2 generation, alpha-granule protein secretion (ELISA), and protein phosphorylation from different signaling pathways (immunoblotting) were studied. Results Preincubation of platelets at temperatures higher than 37°C (38.5°–42°C) inhibited thrombin-induced haemostasis including platelet adhesion, aggregation, ATP release, and thromboxane A2 generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release were completely inhibited by hyperthermia, whereas von Willebrand factor (vWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation as well as IkappaB degradation were decreased in a temperature-dependent fashion. Conclusions Higher temperatures, such as those observed with fever or tissue invasion, inhibit the haemostatic functions of platelets and selectively regulate the release of alpha-granule proteins. PMID:21649851

  20. 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

  1. Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

    PubMed Central

    Aslan, Joseph E.; Healy, Laura D.; Wallisch, Michael; Thierheimer, Marisa L. D.; Loren, Cassandra P.; Pang, Jiaqing; Hinds, Monica T.; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding. PMID:26659727

  2. The Effect of Hyperparathyroid State on Platelet Functions and Bone Loss

    PubMed Central

    Yorulmaz, Göknur; Akalın, Aysen; Akay, Olga Meltem; Şahin, Garip; Bal, Cengiz

    2016-01-01

    Objective: Coagulation and fibrinolysis defects were reported in primary hyperparathyroid patients. However, there are not enough data regarding platelet functions in this group of patients. Our aim was to evaluate the platelet functions in primary and secondary hyperparathyroid patients and to compare them with healthy subjects. Materials and Methods: In our study 25 subjects with primary hyperparathyroidism (PHPT), 25 subjects with secondary hyperparathyroidism (SHPT), and 25 healthy controls were included. Platelet functions of the subjects were evaluated by using platelet-rich plasma and platelet aggregation tests induced with epinephrine, adenosine diphosphate (ADP), collagen, and ristocetin. Serum P selectin levels, which indicate platelet activation level, were measured in all subjects. Bone mineral densitometry was performed for all patients. Results: There was no significant difference between the groups with PHPT and SHPT and the control group regarding the platelet aggregation tests and serum P selectin levels. There was also no significant correlation between parathormone levels and aggregation parameters (ristocetin, epinephrine, collagen, and ADP: respectively p=0.446, 0.537, 0.346, and 0.302) and between P selectin (p=0.516) levels. When we separated the patients according to serum calcium levels, there was also no significant difference between aggregation parameters and serum P selectin levels between the patients with hypercalcemia and the patients with normocalcemia. We could not find any significant correlation between aggregation parameters, P selectin levels, and serum calcium levels in this group of patients. Bone loss was greater in patients with PHPT. Conclusion: There is no significant effect of PHPT or SHPT and serum calcium levels on platelet functions when evaluated by aggregation tests. PMID:26377856

  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.

  4. Interaction of a Monoclonal Antibody to Glycoprotein IV (CD36) with Human Platelets and its Effect on Platelet Function.

    PubMed

    Legrand, C; Pidard, D; Beiso, P; Tenza, D; Edelman, L

    1991-01-01

    FA6-152, a monoclonal antibody to platelet membrane glycoprotein IV (CP IV), was used to quantify the expression of this glycoprotein on platelets, as well as to evaluate its role in platelet aggregation. On resting platelets, 19 400 ± 7700 molecules of the (125)I-labelled IgC could bind per platelet (n = 20). Binding was not modified following stimulation of the platelets with ADP (10 µmol/l) or thrombin (0.1 U/ml). Fab fragments prepared from the antibody by papain digestion also bound to the platelet surface in a saturable manner. Both the intact IgC and its Fab fragments were found to inhibit platelet aggregation and secretion induced by ADP or collagen in platelet-rich plasma and by thrombin in platelet suspensions. Under nonstirred conditions, whereby the release reaction was only minimally affected, the antibody markedly inhibited thrombin-induced surface expression of α-granule thrombospondin (TSP), whereas it did not alter the concomitant expression of α-granule fibrinogen. In addition, electron microscopy revealed a predominant distribution of TSP and T;P IV on pseudopodia and between adherent cells on thrombin-stimulated platelets. These findings thus support the hypothesis that the interaction of TSP with GP IV on the platelet surface is required for an optimal platelet aggregation/secretion process to occur.

  5. Caspase-9 mediates the apoptotic death of megakaryocytes and platelets, but is dispensable for their generation and function.

    PubMed

    White, Michael J; Schoenwaelder, Simone M; Josefsson, Emma C; Jarman, Kate E; Henley, Katya J; James, Chloé; Debrincat, Marlyse A; Jackson, Shaun P; Huang, David C S; Kile, Benjamin T

    2012-05-03

    Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.

  6. Recovery Time of Platelet Function After Aspirin Withdrawal

    PubMed Central

    Lee, Jeonghun; Kim, Jeong Kyung; Kim, Jeong Hee; Dunuu, Tsagaan; Park, Sang-Ho; Park, Sang Joon; Kang, Ji Yeon; Choi, Rak Kyeong; Hyon, Min Su

    2014-01-01

    Introduction Inappropriate antiplatelet therapy discontinuation increases the risk of thrombotic complications and bleeding after dental procedures. To determine the platelet reactivity recovery time after aspirin withdrawal in vivo, our study was conducted in patients with low-risk cardiovascular disease who can stop aspirin administration following the guidelines stipulated by the American College of Chest Physicians. The time it takes for platelet activity to normalize and the diagnostic accuracy of testing methods were assessed for a residual antiplatelet activity with multiple electrode aggregometry. Our study included patients with clinically indicated hypertension preparing for a dental extraction procedure. Materials and methods A total of 212 patients not taking aspirin (control group) and 248 patients with hypertension receiving long-time aspirin treatment at a 100-mg daily dose were prospectively included in the study, which involved stopping aspirin intake before dental extraction. The residual platelet activity and dental bleeding in patients who stopped aspirin intake were analyzed and compared with those of the control group. In addition, platelet reactivity recovery time and bleeding risk in patients who stopped taking aspirin every 24 hours for 0 to 5 days (0–143 hours) before dental extraction was also assessed. Results Platelet reactivity normalized 96 hours after aspirin withdrawal. The cut-off value of 49 arbitrary units in the arachidonic acid platelet aggregation test excluded the effect of aspirin with 91% sensitivity and 66% specificity. AUC showed 0.86 (P < 0.001) diagnostic accuracy. The immediate bleeding complications in all treatment groups were similar to those seen in the control group and were successfully managed with local hemostatic measures. Conclusions The antiplatelet effects of aspirin disappeared 96 hours after aspirin withdrawal in our study, and dental extractions may be safely performed in this period when appropriate

  7. 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.

  8. Evaluation of blood platelet count and function in patients with erectile dysfunction.

    PubMed

    Aldemir, M; Akdemir, F; Okulu, E; Ener, K; Ozayar, A; Gudeloglu, A

    2016-03-01

    We evaluated and compared blood total platelet count, mean platelet volume (MPV) and platelet distribution width (PDW) values of patients with erectile dysfunction (ED) and control subjects. A total 57 male patients (mean age 49.7 ± 12 years) with ED and 59 control men (mean age 49.7 ± 10.7 years) were included in the study. All patients were evaluated using medical history with International Index of Erectile Function (IIEF) scores, physical examination and routine blood analysis. Total blood count, including white blood cell (WBC), total platelet counts, MPV and PDW parameters, were recorded in both groups. MPV values were detected to be significantly higher in patients with ED than control group: 10.7 ± 1 and 9.72 ± 1.5, respectively (P = 0.001). Similarly, PDW values were significantly higher in patients with ED than control group: 14.6 ± 2.8 and 12.9 ± 1.9, respectively (P = 0.001). However, mean platelet and mean WBC counts were similar in both groups (P = 0.45). We demonstrated that MPV and PDW values significantly increased in patients with ED compared with the control group. According to these findings, platelet function might play an important role in patients with ED that warrants further research.

  9. Survival and function of transfused platelets. Studies in two patients with congenital deficiencies of platelet membrane glycoproteins.

    PubMed

    Cesar, Jesus M; Vecino, Ana M

    2009-05-01

    Platelets of patients suffering from Glanzmann's thrombasthenia (GT) and Bernard Soulier Syndrome (BSS) are defective in different membrane glycoproteins. Since these integrins can be identified by monoclonal antibodies, normal infused platelets could be distinguished from defective platelets and followed by using flow cytometry (FC). We studied this aspect in two recipients suffering, one from GT and the other one, who underwent splenectomy, from BSS. One hour after transfusion, normal platelets comprised 17% of the total platelet population in the patient with GT. Aggregation tests detected a measurable response to collagen (increase of 15% of transmittance). The presence of transfused platelets decreased progressively to 0.8% on day 4, which corresponded with a half-life of 2.6 days. Studies performed in the patient suffering from BSS found that 1 hour after transfusion, 53% of the platelet population corresponded to normal platelets. There was a progressive decay until day 6, which corresponded to a half-life of 4.6 days. Aggregation tests also detected a platelet response to ristocetin from 1 hour after transfusion (47% increase of transmittance) to day 3. FC is useful to measure platelet lifespan in these kinds of patients. We also report the first studies of platelet aggregation after platelet transfusion.

  10. 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.

  11. Evaluation of platelet function using the in vitro bleeding time and corrected count increment of transfused platelets. Comparison between platelet concentrates derived from pooled buffy coates and apheresis.

    PubMed

    Eriksson, L; Kristensen, J; Olsson, K; Bring, J; Högman, C F

    1996-01-01

    The functional capacity of transfused platelets was evaluated with in vitro bleeding time (IVBT) and corrected count increment (CCI) in order to compare platelet concentrates (PCs) derived from pooled buffy coats (BC-PCs) with PCs collected by apheresis (A-PCs). The suspension medium in the BC-PCs was 30% CPD plasma and 70% of an additive solution (containing sodium and potassium chloride, sodium citrate and phosphate, mannitol), and in the A-PCs the medium was 100% CPD plasma. IVBT was evaluated using a Thrombostat 4000/2. BC-PC and A-PC were transfused 57 and 41 times, respectively to 36 patients with chemotherapy-induced thrombocytopenia. PCs transfused within 2 days of donation were considered fresh, and those transfused within 3-5 days were considered stored. IVBT was determined before, as well as 10-30 min and 24 h after transfusion; CCI was determined 10-30 min and 24 h after transfusion. The median pretransfusion IVBT value was 486 s. It was measurable in 21 of 98 (21%) of the transfusions, i.e. below the cutoff limit of 486 s. Ten to 30 min after transfusion, the IVBT showed a measurable reduction in 90% of the transfusions with fresh BC-PCs, 92% of those with fresh a-PCs, 63% of those with stored BC-PCs and 79% of those with stored A-PCs. After 24 h, the corresponding values were 63% for fresh BC-PCs, 50% for fresh A-PCs, 26% for stored BC-PCs and 38% for stored A-PCs. The median value of CCI 10-30 min after transfusion was 20 for fresh BC-PCs, 17 for fresh A-PCs, 16 for stored BC-PCs and 14 for stored A-PCs. The difference in IVBT between fresh and stored BC-PCs was significant (p = 0.032), unlike that between fresh and stored A-PC. After 24 h the corresponding values were 7 for fresh BC-PCs, 4 for fresh A-PCs, 4 for stored BC-PCs and 3 for stored A-PCs. When all transfusions with fresh PCs (BC-PCs + A-PCs) were compared with all transfusions with stored PCs, a statistical difference was demonstrated in both CCI (p = 0.027) and IVBT (p = 0.043). Spearman

  12. Effect of blood nitrite and nitrate levels on murine platelet function.

    PubMed

    Park, Ji Won; Piknova, Barbora; Huang, Paul L; Noguchi, Constance T; Schechter, Alan N

    2013-01-01

    Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO₂⁻) and nitrate (NO₃⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.

  13. Platelet function during cardiopulmonary bypass using multiple electrode aggregometry: comparison of centrifugal and roller pumps.

    PubMed

    Kehara, Hiromu; Takano, Tamaki; Ohashi, Noburo; Terasaki, Takamitsu; Amano, Jun

    2014-11-01

    Blood trauma may be lower with centrifugal pumps (CPs) than with roller pumps (RPs) during cardiopulmonary bypass (CPB), because, unlike RPs, CPs do not compress the tubing, and shear stress is considered lower in CPs than in RPs. However, relative platelet function remains unclear. Using multiple electrode aggregometry (MEA), we compared platelet function with CP and RP. Ten swine underwent CPB for 3 h, with five weaned off using CP and five using RP. Platelet function was measured using MEA, as were hemoglobin concentration and platelet count, before sternotomy, after heparin infusion, 30 min and 3 h after starting CPB, after protamine infusion, and 60 min after stopping CPB. Platelet activation was initiated with adenosine diphosphate (ADP), arachidonic acid (AA), and thrombin receptor-activating protein 6 (TRAP). Fibrinogen, platelet factor 4 (PF4), and β-thromboglobin (β-TG) concentrations were measured before sternotomy and 60 min after stopping CPB. In the CP group and using ADP, aggregation was significantly reduced 30 min (P = 0.019) and 3 h (P = 0.027) after starting CPB, recovering to baseline 60 min after CPB was stopped. In the RP group, aggregation was significantly decreased 30 min (P = 0.007) and 3 h (P = 0.003) after starting CPB and after protamine administration (P = 0.028). With AA, aggregation significantly decreased 30 min after starting CPB in both the CP (P = 0.012) and RP (P = 0.016) groups, slightly increasing 3 h after starting CPB and after protamine infusion, and recovering to baseline 60 min after CPB cessation. With TRAP, aggregation in the CP and RP groups decreased 30 min after starting the pump, although changes were not significant; aggregation gradually recovered after 3 h and returned to baseline 60 min after the pumps were stopped. There were no significant differences at all sampling points of MEA. In both groups, fibrinogen, PF4, and β-TG concentrations were similar 60 min after pump cessation and before sternotomy

  14. 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.

  15. Platelet microparticles: detection and assessment of their paradoxical functional roles in disease and regenerative medicine.

    PubMed

    Burnouf, Thierry; Goubran, Hadi Alphonse; Chou, Ming-Li; Devos, David; Radosevic, Mirjana

    2014-07-01

    There is increasing research on and clinical interest in the physiological role played by platelet microparticles (PMPs). PMPs are 0.1-1-μm fragments shed from plasma membranes of platelets that are undergoing activation, stress, or apoptosis. They have a phospholipid-based structure and express functional receptors from platelet membranes. As they are the most abundant microparticles in the blood and they express the procoagulant phosphatidylserine, PMPs likely complement, if not amplify, the functions of platelets in hemostasis, thrombosis, cancer, and inflammation, but also act as promoters of tissue regeneration. Their size and structure make them instrumental in platelet-cell communications as a delivery tool of platelet-borne bioactive molecules including growth factors, other signaling molecules and micro (mi)RNA. PMPs can therefore be a pathophysiological threat or benefit to the cellular environment when interacting with the blood vasculature. There is also increasing evidence that PMP generation is triggered during blood collection, separation into components, and storage, a phenomenon potentially leading to thrombotic and inflammatory side effects in transfused patients. Evaluating PMPs requires strict pre-analytical and analytical procedures to avoid artifactual generation and ensure accurate assessment of the number, size repartitioning, and functional properties. This review describes the physical and functional methods developed for analyzing and quantifying PMPs. It then presents the functional roles of PMPs as markers or triggers of diseases like thrombosis, atherosclerosis, and cancer, and discusses the possible detrimental immunological impact of their generation in blood components. Finally we review the potential function of PMPs in tissue regeneration and the prospects for their use in therapeutic strategies for human health.

  16. Physical proximity and functional interplay of PECAM-1 with the Fc receptor Fc gamma RIIa on the platelet plasma membrane.

    PubMed

    Thai, Le M; Ashman, Leonie K; Harbour, Stacey N; Hogarth, P Mark; Jackson, Denise E

    2003-11-15

    We and others have recently defined that Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) functions as a negative regulator of platelet-collagen interactions involving the glycoprotein VI/Fc receptor gamma chain (GPVI/FcR-gamma chain) signaling pathway.1,2 In this study, we hypothesized that PECAM-1 may be physically and functionally associated with Fc gamma RIIa on the platelet membrane. The functional relationship between PECAM-1 and Fc gamma RIIa was assessed by determining the effect of anti-PECAM-1 monoclonal antibody Fab fragments on Fc gamma RIIa-mediated platelet aggregation and heparin-induced thrombocytopenia (HITS)-mediated platelet aggregation. Preincubation of washed platelets with monoclonal antibody fragments of 2BD4 directed against PECAM-1 and IV.3 directed against Fc gamma RIIa completely blocked Fc gamma RIIa-mediated platelet aggregation and HITS-mediated platelet aggregation, whereas anti-CD151 antibody had no blocking effect. Coengagement of Fc gamma RIIa and PECAM-1 resulted in negative regulation of Fc gamma RIIa-mediated phospholipase C gamma 2 activation, calcium mobilization, and phosphoinositide 3-kinase-dependent signaling pathways. In addition, the physical proximity of Fc gamma RIIa and PECAM-1 was confirmed by using fluorescence resonance energy transfer and coimmunoprecipitation studies. These results indicate that PECAM-1 and Fc gamma RIIa are colocalized on the platelet membrane and PECAM-1 down-regulates Fc gamma RIIa-mediated platelet responses.

  17. Bcl-xL-inhibitory BH3 mimetics can induce a transient thrombocytopathy that undermines the hemostatic function of platelets.

    PubMed

    Schoenwaelder, Simone M; Jarman, Kate E; Gardiner, Elizabeth E; Hua, My; Qiao, Jianlin; White, Michael J; Josefsson, Emma C; Alwis, Imala; Ono, Akiko; Willcox, Abbey; Andrews, Robert K; Mason, Kylie D; Salem, Hatem H; Huang, David C S; Kile, Benjamin T; Roberts, Andrew W; Jackson, Shaun P

    2011-08-11

    BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)β(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.

  18. Activation of the platelet collagen receptor integrin alpha(2)beta(1): its mechanism and participation in the physiological functions of platelets.

    PubMed

    Jung, S M; Moroi, M

    2000-10-01

    When platelets are stimulated by agonists, integrin alpha(2)beta(1) (GP Ia/IIa), one of the platelet collagen receptors, is activated to forms with high affinities for its ligand collagen. Here we describe our studies to characterize the binding kinetics of the activated integrin forms and the activation mechanism. Under low agonist concentrations, integrin alpha(2)beta(1) is activated through a mechanism involving ADP/ADP receptors; and under high agonist concentrations, multiple signaling pathways are involved in its activation. Such differences in mechanism at low and high agonist concentrations are also suggested in the activation of integrin alpha(IIb)beta(3), the platelet fibrinogen receptor. We describe our flow adhesion studies, from which evidence was obtained about the involvement of integrin alpha(2)beta(1) activation in the physiological function of platelets, adhesion and thrombus formation.

  19. Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.

    PubMed

    Finley, Matthew J; Rauova, Lubica; Alferiev, Ivan S; Weisel, John W; Levy, Robert J; Stachelek, Stanley J

    2012-08-01

    CD47 is a ubiquitously expressed transmembrane protein that, through signaling mechanisms mediated by signal regulatory protein alpha (SIRPα1), functions as a biological marker of 'self-recognition'. We showed previously that inflammatory cell attachment to polymeric surfaces is inhibited by the attachment of biotinylated recombinant CD47 (CD47B). We test herein the hypothesis that CD47 modified blood conduits can reduce platelet and neutrophil activation under clinically relevant conditions. We appended a poly-lysine tag to the C-terminus of recombinant CD47 (CD47L) allowing for covalent linkage to the polymer. SIRPα1 expression was confirmed in isolated platelets. We then compared biocompatibility between CD47B and CD47L functionalized polyvinyl chloride (PVC) surfaces and unmodified control PVC surfaces. Quantitative and Qualitative analysis of blood cell attachment to CD47B and CD47L surfaces, via scanning electron microscopy, showed strikingly fewer platelets attached to CD47 modified surfaces compared to control. Flow cytometry analysis showed that activation markers for neutrophils (CD62L) and platelets (CD62P) exposed to CD47 modified PVC were equivalent to freshly acquired control blood, while significantly elevated in the unmodified PVC tubing. In addition, ethylene oxide gas sterilization did not inhibit the efficacy of the CD47 modification. In conclusion, CD47 modified PVC inhibits both the adhesion and activation of platelets and neutrophils.

  20. [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.

  1. Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

    PubMed Central

    Haas, Jessica; Sandrock-Lang, Kirstin; Gärtner, Florian; Jung, Christian Billy; Zieger, Barbara; Parrotta, Elvira; Kurnik, Karin; Sinnecker, Daniel; Wanner, Gerhard; Laugwitz, Karl-Ludwig; Massberg, Steffen; Moretti, Alessandra

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application. PMID:25607928

  2. Glycoxidized HDL, HDL enriched with oxidized phospholipids and HDL from diabetic patients inhibit platelet function

    PubMed Central

    Lê, Quang Huy; El Alaoui, Meddy; Véricel, Evelyne; Ségrestin, Bérénice; Soulère, Laurent; Guichardant, Michel; Lagarde, Michel; Moulin, Philippe; Calzada, Catherine

    2015-01-01

    Context High-density lipoproteins (HDL) possess atheroprotective properties including anti-thrombotic and antioxidant effects. Very few studies relate to the functional effects of oxidized HDL on platelets in type 2 diabetes (T2D). Objective The objective of our study was to investigate the effects of in vitro glycoxidized HDL, and HDL from T2D patients on platelet aggregation and arachidonic acid signaling cascade. At the same time, the contents of hydroxylated fatty acids were assessed in HDL. Results Compared to control HDL, in vitro glycoxidized HDL had decreased proportions of linoleic (LA) and arachidonic (AA) acids in phospholipids and cholesteryl esters, and increased concentrations of hydroxy-octadecadienoic acids (9-HODE and 13-HODE) and 15-hydroxy-eicosatetraenoic acid (15-HETE), derived from LA and AA respectively, especially hydroxy derivatives esterified in phospholipids. Glycoxidized HDL dose-dependently decreased collagen-induced platelet aggregation by binding to SR-BI. Glycoxidized HDL prevented collagen-induced increased phosphorylation of platelet p38 MAPK and cytosolic phospholipase A2, as well as intracellular calcium mobilization. HDL enriched with oxidized phospholipids, namely PC(16:0/13-HODE) dose-dependently inhibited platelet aggregation. Increased concentrations of 9-HODE, 13-HODE and 15-HETE in phospholipids (2.1, 2.1 and 2.4-fold increase respectively) were found in HDL from patients with T2D, and these HDL also inhibited platelet aggregation via SR-BI. Conclusions Altogether, our results indicate that in vitro glycoxidized HDL as well as HDL from T2D patients inhibit platelet aggregation, and suggest that oxidized LA-containing phospholipids may contribute to the anti-aggregatory effects of glycoxidized HDL and HDL from T2D patients. PMID:25794249

  3. Inhibition of platelet function: does it offer a chance of better cancer progression control?

    PubMed

    Sierko, Ewa; Wojtukiewicz, Marek Z

    2007-10-01

    Thrombocytosis is frequently (10 to 57%) observed in cancer patients. Although the mechanisms underlying thrombocytosis are not yet fully elucidated, tumor-derived factors with thrombopoietin-like activity, growth factors, platelet-derived microparticles, and factors released from bone marrow endothelial cells as well as growth factors secreted by megakaryocytes (acting via an autocrine loop) are claimed to influence this process. The course of cancer is strongly associated with hypercoagulable state, which results from direct influences of tumor cells themselves and various indirect mechanisms. Activated platelets provide procoagulant surface amplifying the coagulation process. It is well documented that proteins of the hemostatic system influence different steps of metastasis, angiogenesis, and proteolytic events. Much less is known about the role of platelets in tumor growth and their possible contribution to prevention of tumor cells from the host immune system. Multidirectional activities of platelets during tumor development and metastatic dissemination create a possibility of introducing antiplatelet agents in anticancer therapy. The spectrum of plausible therapies includes antibodies against glycoprotein IIb-IIIa, direct thrombin inhibitors, protease activated receptor-1 targeted therapy, as well as cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors. However, there is no sufficient information on a specific type of cancer where progression does depend on platelet function. Despite numerous experimental studies conducted, to date none of the new specific antiplatelet agents were tested in clinical trials in a cancer patient population.

  4. Human blood platelets lack nitric oxide synthase activity.

    PubMed

    Böhmer, Anke; Gambaryan, Stepan; Tsikas, Dimitrios

    2015-01-01

    Reports on expression and functionality of nitric oxide synthase (NOS) activity in human blood platelets and erythrocytes are contradictory. We used a specific gas chromatography-mass spectrometry (GC-MS) method to detect NOS activity in human platelets. The method measures simultaneously [(15)N]nitrite and [(15)N]nitrate formed from oxidized (15)N-labeled nitric oxide ((15)NO) upon its NOS-catalyzed formation from the substrate l-[guanidino-(15)N2]-arginine. Using this GC-MS assay, we did not detect functional NOS in non-stimulated platelets and in intact platelets activated by various agonists (adenosine diphosphate, collagen, thrombin, or von Willebrand factor) or lysed platelets. l-[guanidino-nitro]-Arginine-inhibitable NOS activity was measured after addition of recombinant human endothelial NOS to lysed platelets. Previous and recent studies from our group challenge expression and functionality of NOS in human platelets and erythrocytes.

  5. 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

  6. Effect of varying nitric oxide release to prevent platelet consumption and preserve platelet function in an in vivo model of extracorporeal circulation.

    PubMed

    Skrzypchak, Amy M; Lafayette, Nathan G; Bartlett, Robert H; Zhou, Zhengrong; Frost, Megan C; Meyerhoff, Mark E; Reynolds, Melissa M; Annich, Gail M

    2007-05-01

    The gold standard for anticoagulation during extracorporeal circulation (ECC) remains systemic heparinization and the concomitant risk of bleeding in an already critically ill patient could lead to death. Normal endothelium is a unique surface that prevents thrombosis by the release of antiplatelet and antithrombin agents. Nitric oxide (NO) is one of the most potent, reversible antiplatelet agents released from the endothelium. Nitric oxide released from within a polymer matrix has been proven effective for preventing platelet activation and adhesion onto extracorporeal circuits. However, the critical NO release (NO flux) threshold for thrombus prevention during ECC has not yet been determined. Using a 4-hour arteriovenous (AV) rabbit model of ECC, we sought to find this threshold value for ECC circuits, using an improved NO-releasing coating (Norel-b). Four groups of animals were tested at variable NO flux levels. Hourly blood samples were obtained for measurement of arterial blood gases, platelet counts, fibrinogen levels and platelet function (via aggregometry). A custom-built AV circuit was constructed with 36 cm of poly(vinyl)chloride (PVC) tubing, a 14 gauge (GA) angiocatheter for arterial access and a modified 10 French (Fr) thoracic catheter for venous access. The Norel-b coating reduced platelet activation and thrombus formation, and preserved platelet function - in all circuits that exhibited an NO flux of 13.65 x 10(10) mol x cm(-2) x min(-1). These results were significant when compared with the controls. With the Norel-b coating, the NO flux from the extracorporeal circuit surface can be precisely controlled by the composition of the polymer coating used, and such coatings are shown to prevent platelet consumption and thrombus formation while preserving platelet function in the animal.

  7. 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.

  8. CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

    PubMed

    Boiret, N; Kanold, J; Fouassier, M; Bons, J M; Halle, P; Rapatel, C; Berger, J; Pireyre, P; Blanzat, V; Travade, P; Bonhomme, J; Demeocq, F; Berger, M G

    2000-08-01

    Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

  9. Performance of the platelet function analyser PFA-100 in testing abnormalities of primary haemostasis.

    PubMed

    Harrison, P; Robinson, M S; Mackie, I J; Joseph, J; McDonald, S J; Liesner, R; Savidge, G F; Pasi, J; Machin, S J

    1999-01-01

    The PFA-100 device is a new instrument for the in-vitro testing of platelet function. Primary haemostasis is stimulated by recording the closure time taken for platelets to seal a 150 microm aperture in the centre of a membrane coated with collagen and either epinephrine or ADP. Patients with type 3 von Willebrand's disease (n = 4) all had infinitely prolonged closure times (> 200 s) with both types of cartridge. A patient with afibrinogenemia exhibited only slightly prolonged closure times of 111 and 166 s for the ADP and epinephrine membranes, respectively. Patients with Glanzmann's thrombasthenia (n = 6) and Bernard Soulier syndrome (n = 2) had grossly prolonged closure times (> 200 s) with both types of cartridges. These results confirmed that the PFA-100 system was highly dependent on normal von Willebrand factor, glycoprotein Ib and glycoprotein IIb/IIIa levels but not on plasma fibrinogen. Patients with storage pool disease (n = 6) and Hermansky Pudlak syndrome (n = 7) had prolonged closure times with the epinephrine cartridge. There was no evidence of enhanced platelet function in patients with antiphospholipid syndrome, in sickle-cell disease or thalassemia. However, ingestion of aspirin resulted in a near consistent and significant prolongation of the closure time for the epinephrine cartridge but not for the ADP cartridge in both normal subjects and patients. The test offers a reliable, reproducible, rapid and simple means of assessing high-shear platelet function in vitro.

  10. 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

  11. Platelet function alterations and their relation to P-selectin (CD62P) expression in children with iron deficiency anemia.

    PubMed

    Yıldırım, Zuhal K; Orhan, Mehmet F; Büyükavcı, Mustafa

    2011-03-01

    Iron deficiency anemia (IDA) may cause platelet aggregation dysfunction and this can be reversed by iron therapy. On the other hand, it has been reported that the platelet fractions carrying the platelet activation markers, CD62P and CD63, are increased in thalassemic patients and there is a significant correlation between the increased levels of soluble P-selectin and free iron in sickle cell disease. This study was performed to investigate the alterations of platelet functions and whether iron deficiency results in diminished expression of activation marker (P-selectin; CD62P) leading to platelet aggregation dysfunction in children with IDA. Hemoglobin, erythrocyte indices (mean erythrocyte volume and red blood cell distribution width), serum levels of iron, transferrin and ferritin, platelet aggregation tests (with ADP, collagen, and ristocetin), PFA-100 closure time, and CD62P expression were evaluated in fasting blood samples of 22 children with IDA and 20 children without anemia. CD62P expression was detected by flow cytometry in normal and 5 μmol/l ADP-activated platelets. Mean closure times were longer in the patient group than control. In platelet aggregation tests, mean values of maximum aggregation times by ristocetin, ADP, and collagen were also more prolonged in patient group. Ristocetin-induced maximum aggregation rates (amplitude) were significantly higher in patients. However, ADP and collagen induction did not produce the same effect. CD62P expressions were significantly higher on activated platelets of the patient group, although they were similar in both groups before activation by ADP. These findings suggest that platelet aggregation and adhesion have been delayed in children with IDA; however, platelet function abnormalities are not associated with CD62P expression on platelet surface.

  12. Mechanism of activation and functional role of protein kinase Ceta in human platelets.

    PubMed

    Bynagari, Yamini S; Nagy, Bela; Tuluc, Florin; Bhavaraju, Kamala; Kim, Soochong; Vijayan, K Vinod; Kunapuli, Satya P

    2009-05-15

    The novel class of protein kinase C (nPKC) isoform eta is expressed in platelets, but not much is known about its activation and function. In this study, we investigated the mechanism of activation and functional implications of nPKCeta using pharmacological and gene knock-out approaches. nPKCeta was phosphorylated (at Thr-512) in a time- and concentration-dependent manner by 2MeSADP. Pretreatment of platelets with MRS-2179, a P2Y1 receptor antagonist, or YM-254890, a G(q) blocker, abolished 2MeSADP-induced phosphorylation of nPKCeta. Similarly, ADP failed to activate nPKCeta in platelets isolated from P2Y1 and G(q) knock-out mice. However, pretreatment of platelets with P2Y12 receptor antagonist, AR-C69331MX did not interfere with ADP-induced nPKCeta phosphorylation. In addition, when platelets were activated with 2MeSADP under stirring conditions, although nPKCeta was phosphorylated within 30 s by ADP receptors, it was also dephosphorylated by activated integrin alpha(IIb)beta3 mediated outside-in signaling. Moreover, in the presence of SC-57101, a alpha(IIb)beta3 receptor antagonist, nPKCeta dephosphorylation was inhibited. Furthermore, in murine platelets lacking PP1cgamma, a catalytic subunit of serine/threonine phosphatase, alpha(IIb)beta3 failed to dephosphorylate nPKCeta. Thus, we conclude that ADP activates nPKCeta via P2Y1 receptor and is subsequently dephosphorylated by PP1gamma phosphatase activated by alpha(IIb)beta3 integrin. In addition, pretreatment of platelets with eta-RACK antagonistic peptides, a specific inhibitor of nPKCeta, inhibited ADP-induced thromboxane generation. However, these peptides had no affect on ADP-induced aggregation when thromboxane generation was blocked. In summary, nPKCeta positively regulates agonist-induced thromboxane generation with no effects on platelet aggregation.

  13. Functional self-association of von Willebrand factor during platelet adhesion under flow

    NASA Astrophysics Data System (ADS)

    Savage, Brian; Sixma, Jan J.; Ruggeri, Zaverio M.

    2002-01-01

    We have used recombinant wild-type human von Willebrand factor (VWF) and deletion mutants lacking the A1 and A3 domains, as well as specific function-blocking monoclonal antibodies, to demonstrate a functionally relevant self-association at the interface of soluble and surface-bound VWF. Platelets perfused at the wall shear rate of 1,500 s1 over immobilized VWF lacking A1 domain function failed to become tethered to the surface when they were in a plasma-free suspension with erythrocytes, but adhered promptly if soluble VWF with functional A1 domain was added to the cells. The same results were observed when VWF was immobilized onto collagen through its A3 domain and soluble VWF with deleted A3 domain was added to the cells. Thus, VWF bound to glass or collagen sustains a process of homotypic self-association with soluble VWF multimers that, as a result, can mediate platelet adhesion. The latter finding demonstrates that direct immobilization on a substrate is not a strict requirement for VWF binding to platelet glycoprotein Ib. The dynamic and reversible interaction of surface-bound and soluble VWF appears to be specifically homotypic, because immobilized BSA, human fibrinogen, and fibronectin cannot substitute for VWF in the process. Our findings highlight a newly recognized role of circulating VWF in the initiation of platelet adhesion. The self-assembly of VWF multimers on an injured vascular surface may provide a relevant contribution to the arrest of flowing platelets opposing hemodynamic forces, thus facilitating subsequent thrombus growth.

  14. Novel function for blood platelets and podoplanin in developmental separation of blood and lymphatic circulation.

    PubMed

    Uhrin, Pavel; Zaujec, Jan; Breuss, Johannes M; Olcaydu, Damla; Chrenek, Peter; Stockinger, Hannes; Fuertbauer, Elke; Moser, Markus; Haiko, Paula; Fässler, Reinhard; Alitalo, Kari; Binder, Bernd R; Kerjaschki, Dontscho

    2010-05-13

    During embryonic development, lymph sacs form from the cardinal vein, and sprout centrifugally to form mature lymphatic networks. Separation of the lymphatic from the blood circulation by a hitherto unknown mechanism is essential for the homeostatic function of the lymphatic system. O-glycans on the lymphatic endothelium have recently been suggested to be required for establishment and maintenance of distinct blood and lymphatic systems, primarily by mediating proper function of podoplanin. Here, we show that this separation process critically involves platelet activation by podoplanin. We found that platelet aggregates build up in wild-type embryos at the separation zone of podoplanin(+) lymph sacs and cardinal veins, but not in podoplanin(-/-) embryos. Thus, podoplanin(-/-) mice develop a "nonseparation" phenotype, characterized by a blood-filled lymphatic network after approximately embryonic day 13.5, which, however, partially resolves in postnatal mice. The same embryonic phenotype is also induced by treatment of pregnant mice with acetyl salicylic acid, podoplanin-blocking antibodies, or by inactivation of the kindlin-3 gene required for platelet aggregation. Therefore, interaction of endothelial podoplanin of the developing lymph sac with circulating platelets from the cardinal vein is critical for separating the lymphatic from the blood vascular system.

  15. Platelets Cellular and Functional Characteristics in Patients with Atrial Fibrillation: A Comprehensive Meta-Analysis and Systematic Review

    PubMed Central

    Weymann, Alexander; Ali-Hasan-Al-Saegh, Sadeq; Sabashnikov, Anton; Popov, Aron-Frederik; Mirhosseini, Seyed Jalil; Nombela-Franco, Luis; Testa, Luca; Lotfaliani, Mohammadreza; Zeriouh, Mohamed; Liu, Tong; Dehghan, Hamidreza; Yavuz, Senol; de Oliveira Sá, Michel Pompeu Barros; Baker, William L.; Jang, Jae-Sik; Gong, Mengqi; Benedetto, Umberto; Dohmen, Pascal M.; D’Ascenzo, Fabrizio; Deshmukh, Abhishek J.; Biondi-Zoccai, Giuseppe; Calkins, Hugh; Stone, Gregg W.

    2017-01-01

    Background This systematic review with meta-analysis aimed to determine the strength of evidence for evaluating the association of platelet cellular and functional characteristics including platelet count (PC), MPV, platelet distribution width (PDW), platelet factor 4, beta thromboglobulin (BTG), and p-selectin with the occurrence of atrial fibrillation (AF) and consequent stroke. Material/Methods We conducted a meta-analysis of observational studies evaluating platelet characteristics in patients with paroxysmal, persistent and permanent atrial fibrillations. A comprehensive subgroup analysis was performed to explore potential sources of heterogeneity. Results Literature search of all major databases retrieved 1,676 studies. After screening, a total of 73 studies were identified. Pooled analysis showed significant differences in PC (weighted mean difference (WMD)=−26.93 and p<0.001), MPV (WMD=0.61 and p<0.001), PDW (WMD=−0.22 and p=0.002), BTG (WMD=24.69 and p<0.001), PF4 (WMD=4.59 and p<0.001), and p-selectin (WMD=4.90 and p<0.001). Conclusions Platelets play a critical and precipitating role in the occurrence of AF. Whereas distribution width of platelets as well as factors of platelet activity was significantly greater in AF patients compared to SR patients, platelet count was significantly lower in AF patients. PMID:28302997

  16. The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways.

    PubMed

    Burkhart, Julia M; Vaudel, Marc; Gambaryan, Stepan; Radau, Sonja; Walter, Ulrich; Martens, Lennart; Geiger, Jörg; Sickmann, Albert; Zahedi, René P

    2012-10-11

    Antiplatelet treatment is of fundamental importance in combatting functions/dysfunction of platelets in the pathogenesis of cardiovascular and inflammatory diseases. Dysfunction of anucleate platelets is likely to be completely attributable to alterations in posttranslational modifications and protein expression. We therefore examined the proteome of platelets highly purified from fresh blood donations, using elaborate protocols to ensure negligible contamination by leukocytes, erythrocytes, and plasma. Using quantitative mass spectrometry, we created the first comprehensive and quantitative human platelet proteome, comprising almost 4000 unique proteins, estimated copy numbers for ∼ 3700 of those, and assessed intersubject (4 donors) as well as intrasubject (3 different blood samples from 1 donor) variations of the proteome. For the first time, our data allow for a systematic and weighted appraisal of protein networks and pathways in human platelets, and indicate the feasibility of differential and comprehensive proteome analyses from small blood donations. Because 85% of the platelet proteome shows no variation between healthy donors, this study represents the starting point for disease-oriented platelet proteomics. In the near future, comprehensive and quantitative comparisons between normal and well-defined dysfunctional platelets, or between platelets obtained from donors at various stages of chronic cardiovascular and inflammatory diseases will be feasible.

  17. Pharmacodynamic effects of cangrelor and clopidogrel: the platelet function substudy from the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials.

    PubMed

    Angiolillo, Dominick J; Schneider, David J; Bhatt, Deepak L; French, William J; Price, Matthew J; Saucedo, Jorge F; Shaburishvili, Tamaz; Huber, Kurt; Prats, Jayne; Liu, Tiepu; Harrington, Robert A; Becker, Richard C

    2012-07-01

    Cangrelor is an intravenous antagonist of the P2Y(12) receptor characterized by rapid, potent, predictable, and reversible platelet inhibition. However, cangrelor was not superior to clopidogrel in reducing the incidence of ischemic events in the cangrelor versus standard therapy to achieve optimal management of platelet inhibition (CHAMPION) trials. A prospectively designed platelet function substudy was performed in a selected cohort of patients to provide insight into the pharmacodynamic effects of cangrelor, particularly in regard to whether cangrelor therapy may interfere with the inhibitory effects of clopidogrel. This pre-defined substudy was conducted in a subset of patients from the CHAMPION-PCI trial (n = 230) comparing cangrelor with 600 mg of clopidogrel administered before percutaneous coronary intervention (PCI) and from the CHAMPION-PLATFORM trial (n = 4) comparing cangrelor at the time of PCI and 600 mg clopidogrel given after the PCI. Pharmacodynamic measures included P2Y12 reaction units (PRU) assessed by VerifyNow P2Y12 testing (primary endpoint marker), platelet aggregation by light transmittance aggregometry following 5 and 20 μmol/L adenosine diphosphate stimuli, and markers of platelet activation determined by flow cytometry. The primary endpoint was the percentage of patients who achieved <20 % change in PRU between baseline and >10 h after PCI. The main trial was stopped early limiting enrollment in the platelet substudy. A total of 167 patients had valid pharmacodynamic assessments for the primary endpoint. The percent of individuals achieving <20 % change in PRU between baseline and >10 h after PCI was higher with cangrelor + clopidogrel (32/84, 38.1 %) compared with placebo + clopidogrel (21/83, 25.3 %), but this was not statistically significant (difference:12.79 %, 95 % CI: -1.18 %, 26.77 %;p = 0.076). All pharmacodynamic markers as well as the prevalence of patients with high on-treatment platelet reactivity were significantly lower

  18. Influence of low-dose proton pump inhibitors administered concomitantly or separately on the anti-platelet function of clopidogrel.

    PubMed

    Furuta, Takahisa; Sugimoto, Mitsushige; Kodaira, Chise; Nishino, Masafumi; Yamade, Mihoko; Uotani, Takahiro; Sahara, Shu; Ichikawa, Hitomi; Kagami, Takuma; Iwaizumi, Moriya; Hamaya, Yasushi; Osawa, Satoshi; Sugimoto, Ken; Umemura, Kazuo

    2017-04-01

    Proton pump inhibitors (PPIs) at low doses can effectively prevent gastrointestinal bleeding due to aspirin and are widely used in Japan for gastroprotection in patients taking anti-platelet agents. We examined the influence of different PPIs at low doses administered concomitantly or separately on anti-platelet functions of clopidogrel. In 41 healthy Japanese volunteers with different CYP2C19 genotypes who took clopidogrel 75 mg in the morning alone, or with omeprazole 10 mg, esomeprazole 10 mg, lansoprazole 15 mg, or rabeprazole 10 mg, either concomitantly in the morning or separately in the evening, we measured the inhibition of platelet aggregation (IPA, %) using VerifyNow P2Y12 assay at 4 h after the last clopidogrel dose on Day 7 of each regimen. IPA by clopidogrel with rabeprazole administered at lunchtime, approximately 4 h after clopidogrel, was also measured. Mean IPAs in those concomitantly receiving omeprazole, esomeprazole, lansoprazole or rabeprazole (47.2 ± 21.1%, 43.2 ± 20.2%, 46.4 ± 18.8%, and 47.3 ± 19.2%, respectively) were significantly decreased compared with those receiving clopidogrel alone (56.0%) (all ps < 0.001). This decrease was observed when PPIs were administered separately in the evening. However, IPA by clopidogrel with rabeprazole administered at lunchtime was 51.6%, which was markedly similar to that of clopidogrel alone (p = 0.114). All tested PPIs reduce the efficacy of clopidogrel when administered concomitantly. Our preliminary data suggest that administration of rabeprazole 4 h following clopidogrel may minimize potential drug-drug interactions.

  19. Aptamer-functionalized silver nanoparticles for scanometric detection of platelet-derived growth factor-BB.

    PubMed

    Hu, Hongting; Li, Hui; Zhao, Yaju; Dong, Shiyu; Li, Wei; Qiang, Weibing; Xu, Danke

    2014-02-17

    In this work, we reported a scanometric assay system based on the aptamer-functionalized silver nanoparticles (apt-AgNPs) for detection of platelet-derived growth factor-BB (PDGF-BB) protein. The aptamer and ssDNA were bound with silver nanoparticles by self-assembly of sulfhydryl group at 5' end to form the apt-AgNPs probe. The apt-AgNPs probe can catalyze the reduction of metallic ions in color agent to generate metal deposition that can be captured both by human eyes and a flatbed scanner. Two different color agents, silver enhancer solution and color agent 1 (10 mM HAuCl4+2 mM hydroquinone) were used to develop silver and gold shell on the surface of AgNPs separately. The results demonstrated that the formation of Ag core-Au shell structure had some advantages especially in the low concentrations. The apt-AgNPs probe coupled with color agent 1 showed remarkable superiority in both sensitivity and detection limit compared to the apt-AuNPs system. The apt-AgNPs system also produced a wider linear range from 1.56 ng mL(-1) to 100 ng mL(-1) for PDGF-BB with the detection limit lower than 1.56 ng mL(-1). The present strategy was applied to the determination of PDGF-BB in 10% serum, and the results showed that it had good specificity in complex biological media.

  20. Increased platelet reactivity in patients with late-stage metastatic cancer

    PubMed Central

    Cooke, Niamh M; Egan, Karl; McFadden, Siobhan; Grogan, Liam; Breathnach, Oscar S; O'Leary, John; Hennessy, Bryan T; Kenny, Dermot

    2013-01-01

    Abstract Platelet hyperreactivity is associated with an increased risk of thrombosis. Cancer patients are at an increased risk of thrombosis, a risk that increases with disease progression. While cancer patients show evidence of platelet activation in vivo, few studies have extensively assessed whether these patients display platelet hyperreactivity. We hypothesized that patients with metastatic cancer would display platelet hyperreactivity, reflecting their associated high risk of thrombosis. In a cohort of patients with metastatic cancer (n = 13), we assessed platelet function using well-established assays of platelet reactivity (agonist-induced platelet aggregation, spontaneous platelet aggregation, and agonist-induced P-selectin expression). In comparison with healthy controls (n = 10), patients with metastatic cancer displayed global platelet hyperreactivity. Agonist-induced platelet aggregation responses to ADP (adenosine diphosphate), epinephrine, collagen, arachidonic acid, and PAR-1 (protease-activated receptor-1) activating peptide, as well as spontaneous platelet aggregation, were significantly increased in patients with metastatic cancer. Furthermore, agonist-induced platelet P-selectin expression was also significantly increased within the patient cohort. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis. We assessed platelet function in a cohort of patients with metastatic cancer (n = 13) using well-established assays of platelet reactivity. Agonist-induced platelet aggregation and activation in response to platelet agonists, as well as spontaneous platelet aggregation, was significantly increased in cancer patients compared with healthy controls. We demonstrate that patients with metastatic cancer are characterized by global platelet hyperreactivity, a factor that may contribute to their increased risk of thrombosis. PMID

  1. Cyclophilin A is an important mediator of platelet function by regulating integrin αIIbβ3 bidirectional signalling.

    PubMed

    Wang, Lian; Soe, Nwe Nwe; Sowden, Mark; Xu, Yingqian; Modjeski, Kristina; Baskaran, Padmamalini; Kim, Yeonghwan; Smolock, Elaine M; Morrell, Craig N; Berk, Bradford C

    2014-05-05

    Cyclophilin A (CyPA) is an important mediator in cardiovascular diseases. It possesses peptidyl-prolyl cis-trans isomerase activity (PPIase) and chaperone functions, which regulate protein folding, intracellular trafficking and reactive oxygen species (ROS) production. Platelet glycoprotein receptor αIIbβ3 integrin activation is the common pathway for platelet activation. It was our objective to understand the mechanism by which CyPA-regulates αIIbβ3 activation in platelets. Mice deficient for CyPA (CyPA-/-) had prolonged tail bleeding time compared to wild-type (WT) controls despite equivalent platelet numbers. In vitro studies revealed that CyPA-/- platelets exhibited dramatically decreased thrombin-induced platelet aggregation. In vivo, formation of occlusive thrombi following FeCl3 injury was also significantly impaired in CyPA-/- mice compared with WT-controls. Furthermore, CyPA deficiency inhibited flow-induced thrombus formation in vitro. Flow cytometry demonstrated that thrombin-induced ROS production and αIIbβ3 activation were reduced in CyPA-/- platelets. Coimmunoprecipitation studies showed ROS-dependent increased association of CyPA and αIIbβ3. This association was dependent upon the PPIase activity of CyPA. Significantly, fibrinogen-platelet binding, platelet spreading and cytoskeleton reorganisation were also altered in CyPA-/- platelets. Moreover, CyPA deficiency prevented thrombin-induced αIIbβ3 and cytoskeleton association. In conclusion, CyPA is an important mediator in platelet function by regulation of αIIbβ3 bidirectionalsignalling through increased ROS production and facilitating interaction between αIIbβ3 and the cell cytoskeleton.

  2. Structure of von Willebrand factor and its function in platelet adhesion and thrombus formation.

    PubMed

    Ruggeri, Z M

    2001-06-01

    The adhesive protein von Willebrand factor mediates the initiation and progression of thrombus formation at sites of vascular injury. von Willebrand factor is synthesized in endothelial cells and megakaryocytes as a very large polymer composed of identical subunits. In the plasma, it appears as a series of multimers of regularly decreasing molecular mass, from several thousand to 500 kDa. The size of circulating von Willebrand factor multimers is controlled by proteolytic cleavage carried out by a specific protease. The biological functions of von Willebrand factor are exerted through specific domains that interact with extracellular matrix components and cell membrane receptors to promote the initial tethering and adhesion of platelets to subendothelial surfaces, as well as platelet aggregation. Moreover, von Willebrand factor binds the procoagulant co-enzyme, factor VIII, contributing to its stability and, indirectly, to its function in the generation of fibrin. This chapter presents a review of current knowledge on the structure, biosynthesis and functions of von Willebrand factor.

  3. The unique contribution of ion channels to platelet and megakaryocyte function.

    PubMed

    Mahaut-Smith, M P

    2012-09-01

    Ion channels are transmembrane proteins that play ubiquitous roles in cellular homeostasis and activation. In addition to their recognized role in the regulation of ionic permeability and thus membrane potential, some channel proteins possess intrinsic kinase activity, directly interact with integrins or are permeable to molecules up to ≈1000 Da. The small size and anuclear nature of the platelet has often hindered progress in understanding the role of specific ion channels in hemostasis, thrombosis and other platelet-dependent events. However, with the aid of transgenic mice and 'surrogate' patch clamp recordings from primary megakaryocytes, important unique contributions to platelet function have been identified for several classes of ion channel. Examples include ATP-gated P2X1 channels, Orai1 store-operated Ca2+ channels, voltage-gated Kv1.3 channels, AMPA and kainate glutamate receptors and connexin gap junction channels. Furthermore, evidence exists that some ion channels, such as NMDA glutamate receptors, contribute to megakaryocyte development. This review examines the evidence for expression of a range of ion channels in the platelet and its progenitor cell, and highlights the distinct roles that these proteins may play in health and disease.

  4. Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies

    PubMed Central

    Lee, Tzu-Yin; Chang, Chao-Chien; Lu, Wan-Jung; Yen, Ting-Lin; Lin, Kuan-Hung; Geraldine, Pitchairaj; Li, Jiun-Yi; Sheu, Joen-Rong

    2017-01-01

    Honokiol, derived from Magnolia officinalis, has various pharmacological properties. Platelet activation plays a critical role in cardiovascular diseases. Honokiol has been reported to inhibit collagen-stimulated rabbit platelet aggregation. However, detailed further studies on the characteristics and functional activity of honokiol in platelet activation are relatively lacking. In the present study, honokiol specifically inhibited platelet aggregation and Ca+2 ion mobilization stimulated with collagen or convulxin, an agonist of glycoprotein (GP) VI, but not with aggretin, an agonist of integrin α2β1. Honokiol also attenuated the phosphorylation of Lyn, PLCγ2, PKC, MAPKs, and Akt after convulxin stimulation. Honokiol have no cytotoxicity in zebrafish embryos. Honokiol diminished the binding of anti-GP VI (FITC-JAQ1) mAb to human platelets, and it also reduced the coimmunoprecipitation of GP VI-bound Lyn after convulxin stimulation. The surface plasmon resonance results revealed that honokiol binds directly to GP VI, with a KD of 289 μM. Platelet function analysis revealed that honokiol substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation in mice. In conclusion, honokiol acts as a potent antagonist of collagen GP VI in human platelets, and it has therapeutic potential in the prevention of the pathological thrombosis. PMID:28054640

  5. 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

  6. [In vitro platelet production].

    PubMed

    Dunois-Lardé, C; Baruch, D

    2011-04-01

    This review aims at presenting a state of the art on platelet functions, not only in well-characterized hemostasis and thrombosis, but also in various domains such as inflammation, immunity, angiogenesis, source of growth factors, metastasis and vascular remodelling. This multivalent phenotype of platelets suggests new potential applications of platelets. The second objective is to present new advances in platelet formation from megakaryocytes and direct platelet release, as initially shown by our group and more recently by others.

  7. A Cell-Based Assay to Assess Hemichannel Function

    PubMed Central

    Krishnan, Srinivasan; Fiori, Mariana C.; Cuello, Luis G.; Altenberg, Guillermo A.

    2017-01-01

    Activation of connexin hemichannels is involved in the pathophysiology of disorders that include deafness, stroke, and cardiac infarct. This aspect makes hemichannels an attractive therapeutic target. Unfortunately, most available inhibitors are not selective or isoform specific, which hampers their translational application. The absence of a battery of useful inhibitors is due in part to the absence of simple screening assays for the discovery of hemichannel-active drugs. Here, we present an assay that we have recently developed to assess hemichannel function. The assay is based on the expression of functional human connexins in a genetically modified bacterial strain deficient in K+ uptake. These modified cells do not grow in low-K+ medium, but functional expression of connexin hemichannels allows K+ uptake and growth. This cell-growth-based assay is simple, robust, and easily scalable to high-throughput multi-well platforms. PMID:28356896

  8. Adenosine and 2'-deoxyadenosine modified with boron cluster pharmacophores as new classes of human blood platelet function modulators.

    PubMed

    Bednarska, Katarzyna; Olejniczak, Agnieszka B; Wojtczak, Błazej A; Sułowska, Zofia; Leśnikowski, Zbigniew J

    2010-05-03

    Novel types of adenosine and 2'-deoxyadenosine derivatives containing boron clusters at positions C2', N6, or C8 were synthesized. The effect of these modified compounds on platelet function was studied. Modification of adenosine at the C2' position with a para-carborane cluster (C(2)B(10)H(11)) results in efficient inhibition of platelet function, including aggregation, protein secretion, and P-selectin expression induced by thrombin or ADP. These preliminary findings and the new chemistry proposed form the basis for the development of a new class of adenosine analogues that modulate human blood platelet activities.

  9. The effect of platelet lysate fibrinogen on the functionality of MSCs in immunotherapy.

    PubMed

    Copland, Ian B; Garcia, Marco A; Waller, Edmund K; Roback, John D; Galipeau, Jacques

    2013-10-01

    Human platelet lysate (PL) represents an attractive alternative to fetal bovine serum (FBS) for the ex vivo expansion of human mesenchymal stromal cells (MSCs). However, there is controversy whether MSCs propagated in unfractionated PL retain their immunosuppressive properties. Since fibrinogen can be a major component of PL, we hypothesized that the fibrinogen content in PL negatively affects the suppressor function of MSCs. Pools of outdated plateletpheresis products underwent a double freeze-thaw centrifugation and filtration to produce unfractionated platelet lysates (uPL), followed by a temperature controlled clotting procedure to produce a fibrinogen depleted platelet lysate (fdPL). Fibrinogen depletion affected neither the mitogenic properties of PL or growth factor content, however fdPL was less prone to develop precipitate over time. Functionally, fibrinogen interacted directly with MSCs, dose dependently increased IL-6, IL-8 and MCP-1 protein production, and compromised the ability of MSCs to up-regulate indoleamine dioxygenase (IDO), as well as, mitigate T-cell proliferation. Similarly uPL expanded MSCs showed a reduced capability of inducing IDO and suppressing T-cell proliferation compared to FBS expanded MSCs. Replacing uPL with fdPL largely restored the immune modulating effects of MSCs. Together these data suggest that fibrinogen negatively affects the immunomodulatory functions of MSCs and fdPL can serve as non-xenogenic mitogenic supplement for expansion of clinical grade MSCs for immune modulation.

  10. Do we need more research on platelet function in South Asian immigrants?

    PubMed

    Dodani, Sunita

    2010-12-01

    South Asian Immigrants (SAIs) have high rates of coronary artery disease (CAD) and its risk factors. This is alarming as i) SAIs are the 2nd fastest growing Asian immigrant population in the US; ii) CAD prevalence is 2 times higher than in other immigrant populations; iii) at any risk factor level, SAIs are at a greater risk of CAD; and iv) Type 2 diabetes is a major risk factor and associated co-morbidity in SAIs. Although clinical guidelines for CAD prevention recommend risk management based on Framingham Risk Scores (FRS), available data suggest that FRS underestimate CAD risk in SAIs. This review presents an overview of platelet related research in SAIs, its importance in predicting CAD risk and methods of diagnosis. Further we provide discussion on the importance of platelet function assessment for more appropriate stratification of SAIs based on cardiovascular risk.

  11. Platelet mitochondrial function in Parkinson's disease. The Royal Kings and Queens Parkinson Disease Research Group.

    PubMed

    Krige, D; Carroll, M T; Cooper, J M; Marsden, C D; Schapira, A H

    1992-12-01

    There is increasing evidence that defective function of the mitochondrial enzyme NADH CoQ reductase (complex I) is involved not only in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity, but also in idiopathic Parkinson's disease (PD). Complex I deficiency has been identified in PD substantia nigra and appears to be disease-specific and selective for the substantia nigra within the central nervous system. We describe a method for preparation of an enriched mitochondrial fraction from 60 mL blood. Using this technique, we analyzed respiratory chain function in 25 patients with PD and 15 matched control subjects. We confirm a previous report of a specific complex I deficiency in PD platelet mitochondria. Although there was a statistically significant decrease in complex I activity in the PD group compared with the control group (p = 0.005), the defect was mild (16%); it was not possible to distinguish PD from control values on an individual basis. This deficiency is not detectable in platelet whole-cell homogenates, presumably reflecting the relative insensitivity of this preparation and the limited decrease in complex I activity in PD. The presence of a mild complex I defect in platelets together with a more severe defect in substantia nigra suggests either that the pharmacological characteristics shared by these two tissues render them susceptible to a particular toxin or toxins, or that the defect is widely distributed and other biochemical events enhance the deficiency in substantia nigra.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Pneumatic tube system transport does not alter platelet function in optical and whole blood aggregometry, prothrombin time, activated partial thromboplastin time, platelet count and fibrinogen in patients on anti-platelet drug therapy

    PubMed Central

    Enko, Dietmar; Mangge, Harald; Münch, Andreas; Niedrist, Tobias; Mahla, Elisabeth; Metzler, Helfried; Prüller, Florian

    2017-01-01

    Introduction The aim of this study was to assess pneumatic tube system (PTS) alteration on platelet function by the light transmission aggregometry (LTA) and whole blood aggregometry (WBA) method, and on the results of platelet count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen. Materials and methods Venous blood was collected into six 4.5 mL VACUETTE® 9NC coagulation sodium citrate 3.8% tubes (Greiner Bio-One International GmbH, Kremsmünster, Austria) from 49 intensive care unit (ICU) patients on dual anti-platelet therapy and immediately hand carried to the central laboratory. Blood samples were divided into 2 Groups: Group 1 samples (N = 49) underwent PTS (4 m/s) transport from the central laboratory to the distant laboratory and back to the central laboratory, whereas Group 2 samples (N = 49) were excluded from PTS forces. In both groups, LTA and WBA stimulated with collagen, adenosine-5’-diphosphate (ADP), arachidonic acid (AA) and thrombin-receptor-activated-peptide 6 (TRAP-6) as well as platelet count, PT, APTT, and fibrinogen were performed. Results No statistically significant differences were observed between blood samples with (Group 1) and without (Group 2) PTS transport (P values from 0.064 – 0.968). The AA-induced LTA (bias: 68.57%) exceeded the bias acceptance limit of ≤ 25%. Conclusions Blood sample transportation with computer controlled PTS in our hospital had no statistically significant effects on platelet aggregation determined in patients with anti-platelet therapy. Although AA induced LTA showed a significant bias, the diagnostic accuracy was not influenced. PMID:28392742

  13. Gender, Race, and Diet Affect Platelet Function Tests in Normal Subjects Contributing to a High Rate of Abnormal Results

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Garrett, Katherine; Stein, Sidney F.

    2015-01-01

    Summary To assess sources of variability in platelet function tests in normal subjects, 64 healthy young adults were tested on 2–6 occasions at 2 week intervals using 4 methods: platelet aggregation (AGG) in platelet-rich plasma (PRP) in the Bio/Data PAP-4 Aggregometer (BD) and Chrono-Log Lumi-Aggregometer (CL); and AGG in whole blood (WB) in the CL and Multiplate Platelet Function Analyzer (MP), with ATP release (REL) in CL-PRP and CL-WB. Food and medication exposures were recorded prospectively for 2 weeks prior to each blood draw. At least one AGG abnormality was seen in 21% of 81 drug-free specimens with CL-PRP, 15% with CL-WB, 13% with BD-PRP, and 6% with MP-WB, increasing with inclusion of REL to 28% for CL-PRP and 30% for CL-WB. Epinephrine AGG and REL were significantly reduced in males (P<0.0001). Ristocetin AGG and collagen and thrombin REL were significantly reduced in Blacks (P<0.0001). One-third of specimens drawn following flavonoid-rich food exposures had aberrant results, compared to 8.5% of specimens without such exposures (P=0.0035). PRP tests had less intra-individual variation than WB tests. Gender, race, diet, and test system affected results of platelet function testing in healthy subjects, suggesting caution when interpreting the results of platelet function testing in patients. PMID:24617520

  14. VerifyNow and VASP phosphorylation assays give similar results for patients receiving clopidogrel, but they do not always correlate with platelet aggregation.

    PubMed

    Bidet, Audrey; Jais, Catherine; Puymirat, Etienne; Coste, Pierre; Nurden, Alan; Jakubowski, Joseph; Nurden, Paquita

    2010-01-01

    Point-of-care testing permits an evaluation of the efficacy of drugs used in the treatment of acute coronary syndromes (ACS). An increased risk of thrombosis after coronary stenting for ACS patients treated with aspirin and clopidogrel has been linked to high platelet reactivity and, for certain patients, poor drug response. The objective of our study was to compare the VerifyNow-P2Y12 device with the VASP (vasodilator-stimulated phosphoprotein) phosphorylation assay and ADP-induced platelet aggregation as assessed by light transmission aggregometry in a group of 81 ACS patients (100 tests) treated in our hospital. There was a good correlation between VerifyNow-P2Y12 and VASP especially during the chronic phase of one month or more after the ischemic event, whereas discordance was sometimes seen with platelet aggregometry. The rapidity and ease of use of the VerifyNow device suggests that it has a valuable place in point-of-care testing of ACS patients.

  15. Alterations of platelet functions in children and adolescents with iron-deficiency anemia and response to therapy.

    PubMed

    Mokhtar, Galila M; Ibrahim, Wafaa E; Kassim, Nevine A; Ragab, Iman A; Saad, Abeer A; Abdel Raheem, Heba G

    2015-01-01

    Several changes in platelets have been reported in patients with iron-deficiency anemia (IDA), so a relationship between iron metabolism and thrombopoiesis should be considered. We aimed to study the alterations of platelet functions in patients with IDA by assessment of platelet aggregation with epinephrine, adenosine diphosphate (ADP) and ristocetin and by measuring platelet function analyzer-100 (PFA-100) closure time together with the effect of iron therapy on the same tests. A follow-up study was conducted in Ain Shams University Children's hospital in the period from June 2011 to June 2012 including 20 patients with confirmed IDA and 20 healthy age- and sex-matched control. Bleeding manifestations were reported. Laboratory analysis included complete blood count, assessment of iron status by measuring serum iron, TIBC and ferritin, assessment of platelet functions by PFA-100 closure time and platelet aggregation with collagen, ADP and ristocetin. Patients with IDA were treated by oral iron therapy 6 mg/kg/day of ferrous sulfate and post-therapeutic re-assessment was done. Mean age of IDA patients was 5.7 ± 4.2 years. Bleeding manifestations were more common in patients group. Mean PFA-100 closure times (with epinephrine) were significantly longer in patients (179.1 ± 86.4 seconds) compared to control group (115 ± 28.5 seconds) (p < 0.05). Platelet aggregation by ADP (38.1 ± 22.2%), epinephrine (19.7 ± 14.2%) and ristocetin (58.8 ± 21.4%) were significantly reduced in patients compared to control (62.7 ± 6.2, 63.3 ± 6.9, 73.8 ± 8.3, respectively; p < 0.001). After treatment platelet aggregation tests induced by ADP (64.78 ± 18.25%), and epinephrine (55.47 ± 24%) were significantly increased in patients with IDA compared to before treatment (39.44 ± 21.85%, 20.33 ± 14.58%; p < 0.001). PFA-100 closure time as well showed significant decreased after treatment (118.4 ± 27.242) compared to before treatment (186.2 ± 90.35; p < 0.05). A negative

  16. Acute impact of conventional and eccentric cycling on platelet and vascular function in patients with chronic heart failure.

    PubMed

    Haynes, Andrew; Linden, Matthew D; Chasland, Lauren C; Nosaka, Kazunori; Maiorana, Andrew J; Dawson, Ellen Adele; Dembo, Lawrence; Naylor, Louise H; Green, Daniel J

    2017-03-16

    Evidence-based guidelines recommend exercise therapy for patients with chronic heart failure (CHF). Such patients have increased atherothrombotic risk. Exercise can transiently increase platelet activation and reactivity and decrease vascular function in healthy participants, although data in CHF is scant. Eccentric (ECC) cycling is a novel exercise modality which may be particularly suited to patients with CHF, but the acute impacts of ECC on platelet and vascular function are currently unknown. Our null hypothesis was that ECC and concentric (CON) cycling, performed at matched external workloads, would not induce changes in platelet or vascular function in patients with CHF. Eleven patients with heart failure with reduced ejection fraction (HFrEF) took part in discrete bouts of ECC and CON cycling. Before and immediately after exercise, vascular function was assessed by measuring diameter and flow mediated dilation (FMD) of the brachial artery. Platelet function was measured by the flow cytometric determination of glycoprotein IIb/IIIa activation and granule exocytosis in the presence and absence of platelet agonists. ECC increased baseline artery diameter (pre: 4.0±0.8mm vs post: 4.2±0.7mm, P=0.04) and decreased FMD%. When changes in baseline artery diameter were accounted for the decrease in FMD post-ECC was no longer significant. No changes were apparent after CON. Neither ECC nor CON resulted in changes to any platelet function measures (all P>0.05). These results suggest both ECC and CON cycling at a moderate intensity and short duration can be performed by patients with HFrEF, without detrimental impacts on vascular or platelet function.

  17. Platelet factor 4 and interleukin-8 CXC chemokine heterodimer formation modulates function at the quaternary structural level.

    PubMed

    Nesmelova, Irina V; Sham, Yuk; Dudek, Arkadiusz Z; van Eijk, Loes I; Wu, Guoping; Slungaard, Arne; Mortari, Frank; Griffioen, Arjan W; Mayo, Kevin H

    2005-02-11

    The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

  18. Measurement of platelet P-selectin for remote testing of platelet function during treatment with clopidogrel and/or aspirin.

    PubMed

    Fox, S C; May, J A; Shah, A; Neubert, U; Heptinstall, S

    2009-06-01

    There is great interest in assessing the efficacy of treatment with clopidogrel and aspirin in patients with cardiovascular disease using procedures that can be used in a remote setting. Here we have established methods to assess the effects of clopidogrel and aspirin on platelets based on measurements of platelet P-selectin. Platelets were stimulated in whole blood by adding the combination of adenosine diphosphate and the TXA(2) mimetic U46619 (ADP/U4, designed to assess P2Y(12) inhibition) or the combination of arachidonic acid and epinephrine (AA/Epi, designed to assess COX-1 inhibition). The stimulated samples were then fixed using a fixative solution that provides stability for at least 9 days, and sent to a central laboratory for analysis of P-selectin by flow cytometry. Measurements were performed in blood from healthy volunteers and patients with cardiovascular disease. The inhibitory effects of clopidogrel and aspirin were assessed ex vivo and the effects of the direct acting P2Y(12) antagonist cangrelor and aspirin were assessed in vitro. Measurements of platelet aggregation were also performed for comparison. In healthy volunteers clopidogrel ex vivo and cangrelor in vitro markedly inhibited P-selectin expression induced by ADP/U4. Aspirin did not inhibit and did not interfere with the effects of clopidogrel or cangrelor using this test. There was very little overlap of results obtained in the absence and presence of clopidogrel or cangrelor. In contrast, over half of 42 patients with cardiovascular disease did not respond well to clopidogrel treatment, although cangrelor was still effective. Aspirin markedly inhibited P-selectin expression induced by AA/Epi. Clopidogrel had much less effect and did not interfere with the effects of aspirin. There was no overlap of results obtained in the absence and presence of aspirin. Aspirin provided near-complete inhibition in 29 of 30 patients with cardiovascular disease. Aggregometry measurements agreed well with

  19. A System for Performing High Throughput Assays of Synaptic Function

    PubMed Central

    Hempel, Chris M.; Sivula, Michael; Levenson, Jonathan M.; Rose, David M.; Li, Bing; Sirianni, Ana C.; Xia, Eva; Ryan, Timothy A.; Gerber, David J.; Cottrell, Jeffrey R.

    2011-01-01

    Unbiased, high-throughput screening has proven invaluable for dissecting complex biological processes. Application of this general approach to synaptic function would have a major impact on neuroscience research and drug discovery. However, existing techniques for studying synaptic physiology are labor intensive and low-throughput. Here, we describe a new high-throughput technology for performing assays of synaptic function in primary neurons cultured in microtiter plates. We show that this system can perform 96 synaptic vesicle cycling assays in parallel with high sensitivity, precision, uniformity, and reproducibility and can detect modulators of presynaptic function. By screening libraries of pharmacologically defined compounds on rat forebrain cultures, we have used this system to identify novel effects of compounds on specific aspects of presynaptic function. As a system for unbiased compound as well as genomic screening, this technology has significant applications for basic neuroscience research and for the discovery of novel, mechanism-based treatments for central nervous system disorders. PMID:21998743

  20. 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

  1. Identification of ITGA2B and ITGB3 Single-Nucleotide Polymorphisms and Their Influences on the Platelet Function

    PubMed Central

    Xiang, Qian; Ji, Shun-Dong; Zhang, Zhuo; Zhao, Xia

    2016-01-01

    The aim of the study was to investigate ITGA2B and ITGB3 genetic polymorphisms and to evaluate the variability in the platelet function in healthy Chinese subjects. The genetic sequence of the entire coding region of the ITGA2B and ITGB3 genes was investigated. Adenosine diphosphate-induced platelet aggregation, glycoprotein IIb/IIIa content, bleeding time, and coagulation indexes were detected. Thirteen variants in the ITGA2B locus and 29 variants in the ITGB3 locus were identified in the Chinese population. The rs1009312 and rs2015049 were associated with the mean platelet volume. The rs70940817 was significantly correlated with the prothrombin time. The rs70940817 and rs112188890 were related with the activated partial thromboplastin time, and ITGB3 rs4642 was correlated with the thrombin time and fibrinogen. The minor alleles of rs56197296 and rs5919 were associated with decreased ADP-induced platelet aggregation, and rs55827077 was related with decreased GPIIb/IIIa per platelet. The rs1009312, rs2015049, rs3760364, rs567581451, rs7208170, and rs117052258 were related with bleeding time. Further studies are needed to explore the clinical importance of ITGA2B and ITGB3 SNPs in the platelet function. PMID:27965976

  2. Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor (RhoGDI) Ly-GDI in platelet function: a spatial systems approach.

    PubMed

    Ngo, Anh T P; Thierheimer, Marisa L D; Babur, Özgün; Rocheleau, Anne D; Huang, Tao; Pang, Jiaqing; Rigg, Rachel A; Mitrugno, Annachiara; Theodorescu, Dan; Burchard, Julja; Nan, Xiaolin; Demir, Emek; McCarty, Owen J T; Aslan, Joseph E

    2017-02-01

    Upon activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42 and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. While RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP)VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

  3. Platelet receptor gain-of-function single nucleotide polymorphisms in carotid and vertebral stenosis patients.

    PubMed

    Lugli, Andrea Kopp; Brown, Martin M; Steffel, Jan; Büchi, Linda; Förnzler, Dorothee; Dupont, Annabelle; Gaussem, Pascale; Forestier, Marc; Beer, Juerg H

    2011-08-01

    The role of platelet receptor gain-of-function single nucleotide polymorphisms (SNP) in cardiovascular disease is controversial. We hypothesised that certain SNPs may accelerate the development of carotid artery stenosis. The intronic PAR-1 receptor intervening sequence-14 A/T (IVSn-14 A/T) polymorphism and three additional platelet receptor polymorphisms, i.e. GPIa (807C/T), GPIbα (5T/C) and HPA-1a/HPA-1b (Pl (A1/A2)) of GPIIIa were studied. The interaction of SNPs with conventional risk factors including male gender, hypertension, high cholesterol, diabetes, advanced age and smoking were investigated. The hypothesis was tested in 114 well-characterised patients with symptomatic carotid or vertebral stenosis from the British CAVATAS population and compared the results with 97 unrelated controls. The allele frequency of the platelet gain-of-function SNP was not significantly different in the CAVATAS population as compared to controls (PAR-1A/T (P = 0.13), GPIa C/T (P = 0.25), GPIIIa HPA-1a/HPA-1b (PlA1/A2) (P = 0.66) and GPIb T/C (P = 0.20)). In the subgroup of smokers, however, the prothrombotic GPIbα C mutated allele was found in a significantly higher frequency in the patient as compared to the control group (P = 0.04). Contrary to the primary hypothesis, the PAR-1A/T SNP as well as the other SNPs tested were not over- or underrepresented in the CAVATAS population. However, a significantly increased prevalence of GPIb-α (5C/T) was found in the subgroup of smokers and may represent an important cofactor in this patient group of our hypothesis-generating study.

  4. Assessment of platelet function in patients receiving tirofiban early after primary coronary intervention

    PubMed Central

    Kupó, Péter; Aradi, Dániel; Tornyos, Adrienn; Tőkés-Füzesi, Margit; Komócsi, András

    2016-01-01

    Background Following percutaneous coronary intervention, combined antiplatelet therapy is necessary. Platelet function testing (PFT) has prognostic value and may be applied in the risk assessment of acute coronary syndrome. In case of combined antiplatelet therapy, PFT may require special laboratory methods, as different antiplatelet agents may influence test results. Materials and methods Platelet functions were measured in stent thrombosis-segment elevation myocardial infarction patients receiving aspirin, clopidogrel, and tirofiban. The first sampling was obtained immediately after the termination of administration of tirofiban. The second sample was drawn at a randomly assigned time between 1 and 6 h. The third sampling was done after a minimum of 24 h of tirofiban cessation. Adenosine diphosphate (ADP)- and thrombin receptor-activating peptide (TRAP)-induced aggregations were measured. Results Thirty-seven patients were included. Both TRAP- and ADP-induced aggregation values were significantly lower immediately after tirofiban termination, than after 24 h [TRAP: 26.41 ± 25.00 units (U) vs. 109.86 ± 23.69 U, p < 0.0001; ADP: 17.43 ± 10.10 U vs. 43.92 ± 23.35 U, p ≤ 0.0001]. Elimination half-life of tirofiban and clopidogrel were 1.34 ± 0.49 and 1.269 ± 0.78, respectively. Conclusion ADP-induced residual platelet reactivity is significantly influenced by the presence of concurrent glycoprotein IIb/IIIa inhibitor. In patients receiving combined antiplatelet treatment, ADP-receptor-specific efficiency measurements are valid only after total elimination of GPIIb/IIIa inhibitors. PMID:28180001

  5. Human Platelets Exhibit Chemotaxis using Functional N-Formyl Peptide Receptors

    DTIC Science & Technology

    2005-01-01

    activated phagocytes. Therefore, we examined the chemotactic migration of platelets qualita- tively by videomicroscopy . Platelets in medium were al- lowed...significantly decreased M. Czapiga et al. /Experimental Hematology 33 (2005) 73–84 79Figure 3. Videomicroscopy of human platelets in response to formyl...selected platelets during videomicroscopy from the time of the addition of fMLF (104 M in 1 µL) or PBS. Movement between markers represents 10 frames

  6. Evaluation of Dried Storage of Platelets for Transfusion: Physiologic Integrity and Hemostatic Functionality

    DTIC Science & Technology

    1993-02-01

    thrombogenic effects of rehydrated platelets involve the use of thrombocytopenic animals. We plan to use a COBE Spectra Blood Cell Separator apheresis ...34AD-A276 018 DT SFEB 2 199-4 SECOND ANNUAL REPORT F 2 4 February 1, 1993 - January 31, 1994 "Evaluation of Dried Storage of Platelets for Transfusion...canine platelets and assessment of multiple infusions of non-labelled rehydrated platelets . The attached subcontract report from Dr. Read and

  7. Alterations of adenine nucleotide metabolism and function of blood platelets in patients with diabetes.

    PubMed

    Michno, Anna; Bielarczyk, Hanna; Pawełczyk, Tadeusz; Jankowska-Kulawy, Agnieszka; Klimaszewska, Joanna; Szutowicz, Andrzej

    2007-02-01

    Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid-reactive species synthesis, and aggregation of platelets from patients with diabetes were 18-82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.

  8. 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

  9. The effects of clopidogrel and omeprazole on platelet function in normal dogs.

    PubMed

    Thames, B E; Lovvorn, J; Papich, M G; Wills, R; Archer, T; Mackin, A; Thomason, J

    2017-04-01

    Omeprazole is used concurrently with clopidogrel to reduce gastrointestinal adverse effects. In humans, the concurrent use of these two drugs can reduce the antiplatelet efficacy of clopidogrel. Our objective was to determine the effects of omeprazole and clopidogrel on platelet function in healthy dogs. A crossover study utilized turbidimetric aggregometry (ADP and collagen) and the PFA-100(®) with the collagen/ADP cartridge to evaluate platelet function in eight healthy dogs during the administration of clopidogrel (1 mg/kg/24 h p.o.), omeprazole (1 mg/kg/24 h p.o.), and a combination of clopidogrel and omeprazole. Drug metabolite concentrations were also measured. Compared to pretreatment, on Days 3 and 5, with ADP as the agonist, there was a significant decrease in maximum amplitude on aggregometry for both clopidogrel and clopidogrel/omeprazole groups. The following revealed no significant differences between clopidogrel and clopidogrel/omeprazole groups when compared on Days 3 and 5: maximum amplitude on aggregometry with ADP or collagen agonists, and PFA-100(®) closure times. When compared to the clopidogrel group, clopidogrel metabolite concentrations in the clopidogrel/omeprazole group were significantly higher on Days 3 and 5. The concurrent administration of omeprazole and clopidogrel in healthy dogs was associated with an increase in the plasma concentration of an inactive metabolite of clopidogrel, but does not significantly alter the antiplatelet effects of clopidogrel.

  10. 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.

  11. Correlation Between the CYP2C19 Phenotype Status and the Results of Three Different Platelet Function Tests in Cardiovascular Disease Patients Receiving Antiplatelet Therapy: An Emphasis on Newly Introduced Platelet Function Analyzer-200 P2Y Test

    PubMed Central

    Li, Shuhua; Choi, Jae-Lim; Guo, Long Zhe; Goh, Ri-Young; Kim, Bo-Ram; Woo, Kwang-Sook; Kim, Moo-Hyun

    2016-01-01

    Background An association has been reported between CYP2C19 polymorphism and the altered antiplatelet activity of clopidogrel. We investigated this association using the newly introduced platelet function analyzer (PFA)-200 (INNOVANCE PFA-200 System; Siemens Healthcare, Germany) P2Y test. Methods Polymorphisms of CYP2C19*2, *3, *17 and the degree of inhibition of platelet function were determined in 83 patients. Three different platelet function tests were used to evaluate the degree of platelet inhibition and to check the association with genotype. Results The post-procedure PFA-200 values of extensive metabolizers (EM) patients (285.3±38.8) were higher than those of intermediate metabolizers (IM) and poor metabolizers (PM) patients (227.7±98.3 and 133.7±99.2, respectively; P=0.024). Light transmittance aggregometry (LTA) and the VerifyNow system showed that the post-procedure values for EM patients were lower than those of IM and PM patients (LTA: 24.4±15.7, 34.1±17.6, and 42.2±16.9, respectively, P<0.001; VerifyNow: 133.2±60.5, 171.5±42.6, and 218.7±59.3, respectively, P<0.001). The high residual platelet reactivity (HPR) rates were significantly different among the EM, IM, and PM groups using PFA-200 (PM:IM:EM=82.4:40.6:11.8, P<0.001). Conclusions Approximately, 59.0% of Korean patients with cardiovascular disease receiving clopidogrel had CYP2C19 loss-of-function genotypes classified as IM or PM, and the frequency was similar to the data from Asian people. The PFA-200, LTA, and VerifyNow platelet function tests revealed evidence of a significant association between the efficacy of clopidogrel and CYP2C19 genotypes. PMID:26522758

  12. Effects of tranexamic acid on platelet function and thrombin generation (ETAPlaT): WOMAN trial sub-study

    PubMed Central

    2016-01-01

    Background. Postpartum haemorrhage (PPH) is a leading cause of maternal death. Tranexamic acid (TXA) has the potential to reduce bleeding and a large randomized placebo controlled trial of its effect in women with PPH (The WOMAN trial) is underway. TXA might also affect coagulation factors and platelets.  Objectives. To examine the effect of TXA on thrombin generation, platelet function, fibrinogen, D-dimer and coagulation factors in women with PPH.  Methods. We will conduct a sub-study within the WOMAN trial. Women with clinically diagnosed primary PPH after vaginal or caesarean delivery are eligible for inclusion. Blood samples will be collected at baseline and 30 minutes after the first dose of study treatment. Using platelet poor plasma we will measure thrombin generation, fibrinogen, D-dimer, factor V and VIII, and Von Willebrand factor. Platelet function will be evaluated in whole blood using Multiplate® tests. Outcomes. The primary outcome is the effect of TXA on thrombin generation. Secondary outcomes include the effect of TXA on platelet function, fibrinogen, D-dimer and coagulation factors. PMID:28090594

  13. Clopidogrel Resistance by P2Y12 Platelet Function Testing in Patients Undergoing Neuroendovascular Procedures

    PubMed Central

    Nordeen, Jerah D.; Patel, Alden V.; Darracott, Robert M.; Johns, Gretchen S.; Taussky, Philipp; Tawk, Rabih G.; Miller, David A.; Freeman, William D.; Hanel, Ricardo A.

    2013-01-01

    Purpose: The purpose of the study was to assess clopidogrel resistance and whether “intensified” antiplatelet therapy guided by platelet inhibition tests during neuroendovascular procedures would reduce ischemic complications. Methods: We conducted a retrospective review of patients at Mayo Clinic in Jacksonville, Florida, who underwent neuroendovascular (NV) procedures and had P2Y12 platelet function testing from October 1, 2009, to September 30, 2010. The primary end-point was to determine P2Y12 resistance to antiplatelet therapy in patients who underwent NV procedures. Secondary objectives included incidence of hemorrhagic and ischemic events and a correlation between resistance and genetic CYP2C19 testing. Results: 160 patients underwent P2Y12 platelet function tests. Eighty-one patients (81/160, 50.6%) met inclusion criteria. Platelet function tests identified 64 patients (79%) as non-resistant (≥20% P2Y12 inhibition) and 17 (21%) as resistant (<20% inhibition) after initial clopidogrel loading. There was an increased rate of death when a complication occurred in the resistant group by 30 day (17% versus 3%; p=0.059) and 90 day follow-up (23% versus 4%; p=0.032). There was no significant association found between complication and loading dose (p=0.0721). Conclusions: 21% of patients undergoing NV procedures were resistant to clopidogrel. Intensifying antiplatelet therapy to achieve ≥20% inhibition on platelet function testing did not result in higher numbers of ischemic or hemorrhagic events, but there was a trend toward more death in the resistant group by 30 and 90 days of those experiencing complication(s). Author Justifications: Jerah D. Nordeen, Pharm.D.: Primary author Alden V. Patel, Pharm.D.: Contributor of professional content, study design Robert M. Darracott, Pharm.D.: Contributor of professional content, study design Gretchen S. Johns, M.D.: Contributor of professional content, study design Philipp Taussky, M.D.: Contributor of professional

  14. 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.

  15. Norepinephrine-mediated regulation of 5HT1 receptor functioning in human platelets.

    PubMed

    Trincavelli, M L; Cuboni, S; Montali, M; Santaguida, S; Lucacchini, A; Martini, C

    2008-07-01

    Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.

  16. High-content live-cell imaging assay used to establish mechanism of trastuzumab emtansine (T-DM1)--mediated inhibition of platelet production.

    PubMed

    Thon, Jonathan N; Devine, Matthew T; Jurak Begonja, Antonija; Tibbitts, Jay; Italiano, Joseph E

    2012-09-06

    Proplatelet production represents a terminal stage of megakaryocyte development during which long, branching processes composed of platelet-sized swellings are extended and released into the surrounding culture. Whereas the cytoskeletal mechanics driving these transformations have been the focus of many studies, significant limitations in our ability to quantify the rate and extent of proplatelet production have restricted the field to qualitative analyses of a limited number of cells over short intervals. A novel high-content, quantitative, live-cell imaging assay using the IncuCyte system (Essen BioScience) was therefore developed to measure the rate and extent of megakaryocyte maturation and proplatelet production under live culture conditions for extended periods of time. As proof of concept, we used this system in the present study to establish a mechanism by which trastuzumab emtansine (T-DM1), an Ab-drug conjugate currently in clinical development for cancer, affects platelet production. High-content analysis of primary cell cultures revealed that T-DM1 is taken up by mouse megakaryocytes, inhibits megakaryocyte differentiation, and disrupts proplatelet formation by inducing abnormal tubulin organization and suppressing microtubule dynamic instability. Defining the pathways by which therapeutics such as T-DM1 affect megakaryocyte differentiation and proplatelet production may yield strategies to manage drug-induced thrombocytopenias.

  17. Platelet function testing during 5-day storage of single and random donor plateletpheresis.

    PubMed

    Akay, O Meltem; Gündüz, Eren; Başyiğit, Hatice; Gulbas, Zafer

    2007-06-01

    Platelet concentrates are routinely manufactured from whole blood by differential centrifugation (random donor platelets-RDP) or by plateletpheresis (single donor platelets-SDP). These platelet concentrates have a storage period of 5 days and many different approaches exist to measure the condition of platelets during their storage. In this study, platelet aggregation testing using adenosine diphosphate (ADP) and collagen and flow cytometric platelet activation analysis using CD41 FITC and CD62 PE before and after ADP was performed on days 1, 3 and 5 of storage of platelet preparations. Thirty three RDPs, stored in Baxter and Kansuk blood bags and 18 SDPs stored in Fresenius blood bags were evaluated. In RDPs and in SDPs; ADP and collagen induced PA responses were decreased significantly on the 3rd and 5th days compared to 1st day. CD62 positive platelet percentage after ADP were decreased significantly on the 3rd and 5th days compared to the 1st day in Kansuk bags. Flow cytometric analysis revealed minor changes in CD41 expression after ADP on the 3rd day compared to 1st day and on the 5th day compared to 3rd day. Differences in CD62 positive platelet percentage were not significant between the RDPs and SDPs. Our results suggest that: (1) ADP and collagen induced PA responses decrease both in RDPs and SDPs during storage. (2) Flow cytometric analysis does not show major significant changes in platelet activation after ADP during storage. (3) Continous shaking on the agitator does not cause a significant change in CD62 positive platelet percentage during storage. (4) Platelet aggregation responses in RDPs stored in Baxter and Kansuk blood bags do not differ during storage.

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

    DTIC Science & Technology

    2015-10-01

    trauma patients. References: 1. Slichter SJ, Harker LA. Preparation and storage of platelet concentrates . II. Storage variables influencing ...Storage variables influencing platelet viability and function. Br J Haematol 1976;34(3):403-419. 2. Becker GA, Tuccelli M, Kunicki T, et al. Studies of...platelet additive solution (PAS) to extend the life of stored platelets. Our project also aims to determine how long acceptable platelet viability can be

  19. Cellular prion protein in blood platelets associates with both lipid rafts and the cytoskeleton.

    PubMed

    Brouckova, Adela; Holada, Karel

    2009-11-01

    The recently shown transmissibility of variant Creutzfeldt-Jakob disease (vCJD) by blood transfusion emphasises the need for better understanding of the cellular prion protein (PrPc) in blood. A substantial amount of cell-associated PrPc in blood resides in platelets. Platelet activation leads to up-regulation of PrPc on the platelet surface and its release on exosomes and microparticles. The sub-cellular localisation and function of platelet PrPc, however, is poorly understood. In the present study, we investigated the association of PrPc with platelet lipid rafts and the platelet cytoskeleton. Immuno-fluorescence microscopy showed that the signals of PrPc and P-selectin, both of which occupy intracellular alpha granules, were separated on the membrane, suggesting organisation in different membrane domains. A flotation assay of platelet lysates demonstrated that a relatively small portion of platelet PrPc floats with lipid rafts, regardless of platelet activation status. This was reversed by depolymerisation of the platelet cytoskeleton, which led to flotation of most platelet PrPc, suggesting that interactions with the cytoskeleton prevent flotation of PrPc rafts. This association of PrPc with the platelet cytoskeleton was confirmed by its presence in both the isolated membrane skeleton and actin cytoskeleton. Platelet activation significantly increased the amount of PrPc associated with the cytoskeleton. Our results indicate that the localisation of PrPc in platelets is complex, with the majority of PrPc present within platelet lipid rafts linked to the platelet cytoskeleton. This localisation places PrPc in a position where it can interact with proteins involved in platelet signalling and eventually with vCJD prions.

  20. Unidirectional transfer of prostaglandin endoperoxides between platelets and endothelial cells.

    PubMed Central

    Schafer, A I; Crawford, D D; Gimbrone, M A

    1984-01-01

    An important determinant of platelet-vessel wall interactions is the local balance of production of endothelial prostacyclin (PGI2) and platelet thromboxane (TX) A2, labile eicosanoids with opposing effects on hemostasis. Disputed evidence suggests that platelet-derived prostaglandin endoperoxide intermediates may be utilized as substrates for vascular PGI2 synthesis. Using several different approaches, we have found that platelets can transfer endoperoxides to cultured endothelial cells for efficient conversion to PGI2, but a reciprocal transfer of endothelial endoperoxides for utilization by platelet thromboxane synthetase does not occur under the same experimental conditions. However, platelets can utilize arachidonic acid released by endothelial cells for lipoxygenase metabolism. We have directly demonstrated the production of [3H]6-keto-PGF1 alpha (the breakdown product of [3H]PGI2) by aspirin-treated endothelial cells in the presence of platelets stimulated with [3H]arachidonic acid. In coincubation experiments using either arachidonate or ionophore A23187 as a stimulus, radioimmunoassay of the net production of arachidonic acid metabolites showed that 6-keto-PGF1 alpha generation by aspirin-treated endothelial cells in the presence of platelets may actually exceed its generation by uninhibited endothelial cells alone. In functional assays, platelet aggregation was inhibited in the presence of aspirin-treated endothelial cells after stimulation with either arachidonate or ionophore A23187. In contrast, the inverse experiments, using aspirin-treated platelets and uninhibited endothelial cells, failed to demonstrate platelet utilization of endothelial endoperoxides for TXA2 production by any of the above methods. These studies thus provide evidence that efficient unidirectional transfer and utilization of platelet-derived endoperoxides for endothelial PGI2 production can occur. This process may serve to amplify PGI2 generation adjacent to areas of vascular

  1. 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.

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. Characterization of a Novel Function-Blocking Antibody Targeted Against The Platelet P2Y1 Receptor

    PubMed Central

    Karim, Zubair A.; Vemana, Hari Priya; Alshbool, Fatima Z.; Lin, Olivia A.; Alshehri, Abdullah M.; Javaherizadeh, Payam; Paez Espinosa, Enma V.; Khasawneh, Fadi T.

    2015-01-01

    Objective Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation, and activates platelets through two G-Protein Coupled Receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor (P2Y12R). While the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. Approach and Results Our goal is to determine whether a novel antibody targeting the ligand binding domain, i.e., second extracellular loop (EL2) of the P2Y1R [abbreviated as EL2Ab] could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and alpha granule secretion and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3 induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose dependent displacement of the radiolabelled P2Y1R antagonist [3H]MRS25000 from its ligand binding site by EL2Ab. Conclusions Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies. PMID:25593131

  7. Evidence that abnormal platelet functions in human Chédiak-Higashi syndrome are the result of a lack of dense bodies.

    PubMed Central

    Rendu, F.; Breton-Gorius, J.; Lebret, M.; Klebanoff, C.; Buriot, D.; Griscelli, C.; Levy-Toledano, S.; Caen, J. P.

    1983-01-01

    The structure and functions of platelets from three patients with the Chédiak-Higashi syndrome were examined. Electron-microscopic observations revealed a large reduction in the number of serotonin-storage granules or dense bodies but otherwise normal ultrastructure and normal amounts of alpha-granules and catalase-positive granules. The number of mepacrine-labeled granules was also reduced. Platelets contained normal amounts of beta-thromboglobulin and Platelet Factor 4. The platelet release reaction studied with thrombin as the inducer was impaired. The serotonin uptake by the patients' platelets was low and not inhibited by reserpine, and its metabolism was increased. These findings clearly show that platelets from human Chédiak-Higashi syndrome are deficient in the storage pool of dense granule substances and suggest that this granule defect has an influence on the release mechanism of other granule constituents. Images Figure 1 Figure 2 Figure 3 PMID:6222656

  8. Statistical analysis plan for the WOMAN-ETAPlaT study: Effect of tranexamic acid on platelet function and thrombin generation

    PubMed Central

    2016-01-01

    Background. Postpartum haemorrhage (PPH) is a potentially life-threatening complication for women, and the leading cause of maternal mortality. Tranexamic acid (TXA) is an antifibrinolytic used worldwide to treat uterine haemorrhage and to reduce blood loss in general surgery. TXA may have effects on thrombin generation, platelet function and coagulation factors as a result of its inhibition on the plasmin. Methods. WOMAN ETAPlaT is a sub-study of the World Maternal Antifibrinolitic trial (WOMAN trial). All adult women clinically diagnosed with PPH after a vaginal delivery or caesarean section, are eligible for inclusion in the study. Blood samples will be collected at the baseline and 30 minutes after the first dose of study treatment is given. Platelet function will be evaluated in whole blood immediately after sampling with Multiplate® tests (ADPtest and TRAPtest). Thrombin generation, fibrinogen, D-dimer, and coagulation factors vW, V and VIII will be analysed using platelet poor plasma. Results. Recruitment to WOMAN ETAPlaT started on 04 November 2013 and closed on 13 January 2015, during this time  188 patients were recruited. The final participant follow-up was completed on 04 March 2015. This article introduces the statistical analysis plan for the study, without reference to unblinded data.   Conclusion. The data from this study will provide evidence for the effect of TXA on thrombin generation, platelet function and coagulation factors in women with PPH. Trial registration: ClinicalTrials.gov Identifier: NCT00872469; ISRCTN76912190

  9. PDK1 selectively phosphorylates Thr(308) on Akt and contributes to human platelet functional responses

    PubMed Central

    Dangelmaier, Carol; Manne, Bhanu Kanth; Liverani, Elizabetta; Jin, Jianguo; Bray, Paul; Kunapuli, Satya P.

    2014-01-01

    Summary 3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt onThr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses. PMID:24352480

  10. A novel role for the fibrinogen Asn-Gly-Arg (NGR) motif in platelet function.

    PubMed

    Moriarty, Róisín; McManus, Ciara A; Lambert, Matthew; Tilley, Thea; Devocelle, Marc; Brennan, Marian; Kerrigan, Steven W; Cox, Dermot

    2015-02-01

    The integrin αIIbβ3 on resting platelets can bind to immobilised fibrinogen resulting in platelet spreading and activation but requires activation to bind to soluble fibrinogen. αIIbβ3 is known to interact with the general integrin-recognition motif RGD (arginine-glycine-aspartate) as well as the fibrinogen-specific γ-chain dodecapeptide; however, it is not known how fibrinogen binding triggers platelet activation. NGR (asparagine-glycine-arginine) is another integrin-recognition sequence present in fibrinogen and this study aims to determine if it plays a role in the interaction between fibrinogen and αIIbβ3. NGR-containing peptides inhibited resting platelet adhesion to fibrinogen with an IC50 of 175 µM but failed to inhibit the adhesion of activated platelets to fibrinogen (IC50> 500 µM). Resting platelet adhesion to mutant fibrinogens lacking the NGR sequences was reduced compared to normal fibrinogen under both static and shear conditions (200 s⁻¹). However, pre-activated platelets were able to fully spread on all types of fibrinogen. Thus, the NGR motif in fibrinogen is the site that is primarily responsible for the interaction with resting αIIbβ3 and is responsible for triggering platelet activation.

  11. Investigation of function similarities between the sarcoplasmic reticulum and platelet calcium-dependent adenosinetriphosphatases with the inhibitors quercetin and calmidazolium

    SciTech Connect

    Fischer, T.H.; Campbell, K.P.; White, G.C. II

    1987-12-01

    The platelet and skeletal sarcoplasmic reticulum calcium-dependent adenosinetriphosphatases (Ca/sup 2 +/-ATPases) were functionally compared with respect to substrate activation by steady-state kinetic methods using the inhibitors quercetin and calmidazolium. Quercetin inhibited platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPase activities in a dose-dependent manner with IC/sub 50/ values of 25 and 10 ..mu..M, respectively. Calmidazolium also inhibited platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPase activities, with half-maximal inhibition measured at 5 and 4 ..mu..M, respectively. Both inhibitors also affected the (/sup 45/Ca) calcium transport activity of intact platelet microsomes at concentrations similar to those which reduced Ca/sup 2 +/-ATPase activity. These inhibitors were then used to examine substrate ligation by the platelet and sarcoplasmic reticulum calcium pump proteins. For both Ca/sup 2 +/-ATPase proteins, quercetin has an affinity for the E-Ca/sub 2/ (fully ligated with respect to calcium at the exterior high-affinity calcium binding sites, unligated with respect to ATP) conformational state of the protein that is approximately 10-fold grater than for other conformational states in the hydrolytic cycle. Quercetin can thus be considered a competitive inhibitor of the calcium pump proteins with respect to ATP. In contrast to the effect of quercetin, calmidazolium interacts with the platelet and sarcoplasmic reticulum Ca/sup 2 +/-ATPases in an uncompetitive manner. The dissociation constants for this inhibitor for the different conformational states of the calcium pump proteins were similar, indicating that calmidazolium has equal affinity for all of the reaction intermediates probed. These observations indicate that the substrate ligation processes are similar for the two pump proteins. This supports the concept that the hydrolytic cycles of the two proteins are comparable.

  12. 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.

  13. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel.

    PubMed

    Dawood, Ban B; Lowe, Gillian C; Lordkipanidzé, Marie; Bem, Danai; Daly, Martina E; Makris, Mike; Mumford, Andrew; Wilde, Jonathan T; Watson, Steve P

    2012-12-13

    Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

  14. Evaluation of coagulation factors and platelet function from an off-line modified ultrafiltration technique for post-cardiopulmonary bypass circuit blood recovery.

    PubMed

    Beckmann, S; Lynn, P; Miller, S; Harris, R; DiMarco, R F; Ross, J E

    2013-05-01

    increased in the final product. The PLT number, although increased by 24%, was not statistically significant. While PLT function assays showed a statistically significant decrease in the levels post-processing, there was substantial platelet function in the MUF product. Overall, the decrease in function was in the range of 10% to 15%. Final product PDGF-αβ and TGF-β1 averaged 11,048 and 2,040 pg/ml, respectively. In these two case series, (ECC) circuit blood concentrated using the HB device showed coagulation studies with significantly lower PT and INR and significantly increased levels of all clotting factors. The findings are similar to trends reported in other studies utilizing conventional MUF and the HB. Functioning platelets remain in the final product, with growth factor concentrations similar to some methods employed to create platelet concentrates to enhance coagulation. Based on the ability of the HB off-line MUF procedure to concentrate circuit blood, the clinical utility of the HB device to decrease allogeneic blood product exposure should be evaluated in a prospective randomized clinical trial.

  15. 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.

  16. Syncytiotrophoblast Extracellular Vesicles from Pre-Eclampsia Placentas Differentially Affect Platelet Function

    PubMed Central

    Tannetta, Dionne S.; Hunt, Kathryn; Jones, Chris I.; Davidson, Naomi; Coxon, Carmen H.; Ferguson, David; Redman, Christopher W.; Gibbins, Jonathan M.; Sargent, Ian L.; Tucker, Katherine L.

    2015-01-01

    Pre-eclampsia (PE) complicates around 3% of all pregnancies and is one of the most common causes of maternal mortality worldwide. The pathophysiology of PE remains unclear however its underlying cause originates from the placenta and manifests as raised blood pressure, proteinuria, vascular or systemic inflammation and hypercoagulation in the mother. Women who develop PE are also at significantly higher risk of subsequently developing cardiovascular (CV) disease. In PE, the failing endoplasmic reticulum, oxidative and inflammatory stressed syncytiotrophoblast layer of the placenta sheds increased numbers of syncytiotrophoblast extracellular vesicles (STBEV) into the maternal circulation. Platelet reactivity, size and concentration are also known to be altered in some women who develop PE, although the underlying reasons for this have not been determined. In this study we show that STBEV from disease free placenta isolated ex vivo by dual placental perfusion associate rapidly with platelets. We provide evidence that STBEV isolated from normal placentas cause platelet activation and that this is increased with STBEV from PE pregnancies. Furthermore, treatment of platelets with aspirin, currently prescribed for women at high risk of PE to reduce platelet aggregation, also inhibits STBEV-induced reversible aggregation of washed platelets. Increased platelet reactivity as a result of exposure to PE placenta derived STBEVs correlates with increased thrombotic risk associated with PE. These observations establish a possible direct link between the clotting disturbances of PE and dysfunction of the placenta, as well as the known increased risk of thromboembolism associated with this condition. PMID:26551971

  17. Nitric oxide released from activated platelets inhibits platelet recruitment.

    PubMed Central

    Freedman, J E; Loscalzo, J; Barnard, M R; Alpert, C; Keaney, J F; Michelson, A D

    1997-01-01

    Vessel injury and thrombus formation are the cause of most ischemic coronary syndromes and, in this setting, activated platelets stimulate platelet recruitment to the growing thrombus. Recently, a constitutive nitric oxide synthase (NOS) has been identified in human platelets. To further define the capacity of platelets to produce nitric oxide (NO), as well as to study the role of this NO in platelet recruitment, we adapted a NO-selective microelectrode for use in a standard platelet aggregometer, thereby permitting simultaneous measurement of platelet aggregation and NO production. Treatment of platelets with the NO synthase inhibitor -NG-nitroarginine methyl ester (L-NAME), reduced NO production by 92+/-8% in response to 5 microM ADP compared to control but increased aggregation by only 15+/-2%. In contrast, L-NAME had a more pronounced effect on platelet recruitment as evidenced by a 35+/-5% increase in the extent of aggregation, a 33+/-3% decrease in cyclic GMP content, and a 31+/-5% increase in serotonin release from a second recruitable population of platelets added to stimulated platelets at the peak of NO production. To study platelet recruitment accurately, we developed an assay that monitors two platelet populations simultaneously. Nonbiotinylated platelets were incubated with L-NAME or vehicle and activated with ADP. At peak NO production, biotinylated platelets were added. As measured by three-color flow cytometry, there was a 56+/-11% increase in the number of P selectin- positive platelets in the nonbiotinylated population treated with L-NAME as compared to control. When biotinylated platelets were added to the L-NAME-treated nonbiotinylated population, the number of P selectin positive biotinylated plate-lets increased by 180+/-32% as compared to biotinylated platelets added to the control. In summary, stimulated platelets produce NO that modestly inhibits platelet activation but markedly inhibits additional platelet recruitment. These data suggest

  18. Clinical value of mean platelet volume for impaired cardiopulmonary function in very old male patients with chronic obstructive pulmonary disease.

    PubMed

    Cui, Hua; Liu, Lin; Wei, Zhimin; Wang, Delong; Hu, Yixin; Hu, Guoliang; Fan, Li

    2012-01-01

    High mean platelet volume (MPV) is a marker of platelet activation. The present study was designed to test if high MPV is associated with impaired cardiopulmonary function in patients with COPD. One hundred and sixteen male outpatients (mean age, 86.03±4.29 years) with COPD were recruited. Blood samples were collected for measurements of MPV and other laboratory data. Lung function and cardiac function were also assessed. Multiple linear regression analyses revealed that MPV was negatively correlated with left ventricular ejection fraction (β=-0.252, p=0.008) and the predicted value of forced expiratory volume in one second (FEV(1)% predicted) (β=-0.384, p=0.0001), whereas MPV was positively correlated with right pulmonary arterial diameter (β=0.311, p=0.005). The present study showed an association between high MPV, a marker of platelet activation, and impaired cardiopulmonary function in elderly COPD male patients. High MPV may be regarded as an early predictive marker of impaired cardiopulmonary function in COPD.

  19. SDF-1α is a novel autocrine activator of platelets operating through its receptor CXCR4.

    PubMed

    Walsh, Tony G; Harper, Matthew T; Poole, Alastair W

    2015-01-01

    Platelets store and secrete the chemokine stromal cell-derived factor (SDF)-1α upon platelet activation, but the ability of platelet-derived SDF-1α to signal in an autocrine/paracrine manner mediating functional platelet responses relevant to thrombosis and haemostasis is unknown. We sought to explore the role of platelet-derived SDF-1α and its receptors, CXCR4 and CXCR7 in facilitating platelet activation and determine the mechanism facilitating SDF-1α-mediated regulation of platelet function. Using human washed platelets, CXCR4 inhibition, but not CXCR7 blockade significantly abrogated collagen-mediated platelet aggregation, dense granule secretion and thromboxane (Tx) A2 production. Time-dependent release of SDF-1α from collagen-activated platelets supports a functional role for SDF-1α in this regard. Using an in vitro whole blood perfusion assay, collagen-induced thrombus formation was substantially reduced with CXCR4 inhibition. In washed platelets, recombinant SDF-1α in the range of 20-100 ng/mL(-1) could significantly enhance platelet aggregation responses to a threshold concentration of collagen. These enhancements were completely dependent on CXCR4, but not CXCR7, which triggered TxA2 production and dense granule secretion. Rises in cAMP were significantly blunted by SDF-1α, which could also enhance collagen-mediated Ca2+ mobilisation, both of which were mediated by CXCR4. This potentiating effect of SDF-1α primarily required TxA2 signalling acting upstream of dense granule secretion, whereas blockade of ADP signalling could only partially attenuate SDF-1α-induced platelet activation. Therefore, this study supports a potentially novel autocrine/paracrine role for platelet-derived SDF-1α during thrombosis and haemostasis, through a predominantly TxA2-dependent and ADP-independent pathway.

  20. A functional CFTR assay using primary cystic fibrosis intestinal organoids.

    PubMed

    Dekkers, Johanna F; Wiegerinck, Caroline L; de Jonge, Hugo R; Bronsveld, Inez; Janssens, Hettie M; de Winter-de Groot, Karin M; Brandsma, Arianne M; de Jong, Nienke W M; Bijvelds, Marcel J C; Scholte, Bob J; Nieuwenhuis, Edward E S; van den Brink, Stieneke; Clevers, Hans; van der Ent, Cornelis K; Middendorp, Sabine; Beekman, Jeffrey M

    2013-07-01

    We recently established conditions allowing for long-term expansion of epithelial organoids from intestine, recapitulating essential features of the in vivo tissue architecture. Here we apply this technology to study primary intestinal organoids of people suffering from cystic fibrosis, a disease caused by mutations in CFTR, encoding cystic fibrosis transmembrane conductance regulator. Forskolin induces rapid swelling of organoids derived from healthy controls or wild-type mice, but this effect is strongly reduced in organoids of subjects with cystic fibrosis or in mice carrying the Cftr F508del mutation and is absent in Cftr-deficient organoids. This pattern is phenocopied by CFTR-specific inhibitors. Forskolin-induced swelling of in vitro-expanded human control and cystic fibrosis organoids corresponds quantitatively with forskolin-induced anion currents in freshly excised ex vivo rectal biopsies. Function of the CFTR F508del mutant protein is restored by incubation at low temperature, as well as by CFTR-restoring compounds. This relatively simple and robust assay will facilitate diagnosis, functional studies, drug development and personalized medicine approaches in cystic fibrosis.

  1. Cbl proteins in platelet activation.

    PubMed

    Buitrago, Lorena; Tsygankov, Alexander; Sanjay, Archana; Kunapuli, Satya P

    2013-01-01

    Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.

  2. Inherited platelet disorders: Insight from platelet genomics using next-generation sequencing

    PubMed Central

    Maclachlan, Annabel; Watson, Steve P.; Morgan, Neil V.

    2017-01-01

    Abstract Inherited platelet disorders (IPDs) are a heterogeneous group of disorders associated with normal or reduced platelet counts and bleeding diatheses of varying severities. The identification of the underlying cause of IPDs is clinically challenging due to the absence of a gold-standard platelet test, and is often based on a clinical presentation and normal values in other hematology assays. As a consequence, a DNA-based approach has a potentially important role in the investigation of these patients. Next-generation sequencing (NGS) technologies are allowing the rapid analysis of genes that have been previously implicated in IPDs or that are known to have a key role in platelet regulation, as well as novel genes that have not been previously implicated in platelet dysfunction. The potential limitations of NGS arise with the interpretation of the sheer volume of genetic information obtained from whole exome sequencing (WES) or whole genome sequencing (WGS) in order to identify function-disrupting variants. Following on from bioinformatic analysis, a number of candidate genetic variants usually remain, therefore adding to the difficulty of phenotype–genotype segregation verification. Linking genetic changes to an underlying bleeding disorder is an ongoing challenge and may not always be feasible due to the multifactorial nature of IPDs. Nevertheless, NGS will play a key role in our understanding of the mechanisms of platelet function and the genetics involved. PMID:27348543

  3. Inherited platelet disorders: Insight from platelet genomics using next-generation sequencing.

    PubMed

    Maclachlan, Annabel; Watson, Steve P; Morgan, Neil V

    2017-01-01

    Inherited platelet disorders (IPDs) are a heterogeneous group of disorders associated with normal or reduced platelet counts and bleeding diatheses of varying severities. The identification of the underlying cause of IPDs is clinically challenging due to the absence of a gold-standard platelet test, and is often based on a clinical presentation and normal values in other hematology assays. As a consequence, a DNA-based approach has a potentially important role in the investigation of these patients. Next-generation sequencing (NGS) technologies are allowing the rapid analysis of genes that have been previously implicated in IPDs or that are known to have a key role in platelet regulation, as well as novel genes that have not been previously implicated in platelet dysfunction. The potential limitations of NGS arise with the interpretation of the sheer volume of genetic information obtained from whole exome sequencing (WES) or whole genome sequencing (WGS) in order to identify function-disrupting variants. Following on from bioinformatic analysis, a number of candidate genetic variants usually remain, therefore adding to the difficulty of phenotype-genotype segregation verification. Linking genetic changes to an underlying bleeding disorder is an ongoing challenge and may not always be feasible due to the multifactorial nature of IPDs. Nevertheless, NGS will play a key role in our understanding of the mechanisms of platelet function and the genetics involved.

  4. Non-Carriers of Reduced-Function CYP2C19 Alleles are Most Susceptible to Impairment of the Anti-Platelet Effect of Clopidogrel by Proton-Pump Inhibitors: A Pilot Study

    PubMed Central

    Lee, Jen-Kuang; Wu, Cho-Kai; Juang, Jyh-Ming; Tsai, Chia-Ti; Hwang, Juey-Jen; Lin, Jiuun-Lee; Chiang, Fu-Tien

    2016-01-01

    Background The phenomenon of CYP2C19 polymorphism affects the metabolism of both clopidogrel and proton-pump inhibitors (PPI). However, concomitant use of both drugs may reduce the desired therapeutic effects. In this study, we evaluated whether individuals with different numbers of reduced-function CYP2C19 alleles were equally affected and whether PPIs with different dependencies on CYP2C19 metabolism were equally involved. Methods Thirty healthy volunteers were recruited to a six-week regimen of clopidogrel. Three PPIs with different metabolic dependencies on CYP2C19 were included and separately administered in this order. Each PPI was given for a week, followed by a one-week washout period before the intervention of the next PPI. The anti-platelet effect was examined by Thromboelastography Platelet MappingTM (TEG®) and vasodilator-stimulated phosphoprotein (VASP) assays. Results Both TEG® and VASP tests showed the same general qualitative trend, but TEG® detected a statistically significant fluctuation of platelet aggregation in response to different drug interventions. The TEG® results also demonstrated that non-carriers experienced the most significant impairment of anti-platelet effect of clopidogrel after concomitant use of PPIs. This impairment was closely related to the metabolic dependence on CYP2C19 of PPI. Conclusions Our study indicated that non-carriers of reduced-function CYP2C19 alleles are most susceptible to impairment of the anti-platelet effect of clopidogrel after concomitant PPI use. Individual subjects are not equally affected, and PPIs are not equally involved. However, large-scale randomized clinical trials are needed to evaluate the clinical outcome. PMID:27122952

  5. A Study of Platelet Inhibition, Using a ‘Point of Care’ Platelet Function Test, following Primary Percutaneous Coronary Intervention for ST-Elevation Myocardial Infarction [PINPOINT-PPCI

    PubMed Central

    Johnson, Thomas W.; Mumford, Andrew D.; Scott, Lauren J.; Mundell, Stuart; Butler, Mark; Strange, Julian W.; Rogers, Chris A.; Reeves, Barnaby C.; Baumbach, Andreas

    2015-01-01

    Background Rapid coronary recanalization following ST-elevation myocardial infarction (STEMI) requires effective anti-platelet and anti-thrombotic therapies. This study tested the impact of door to end of procedure (‘door-to-end’) time and baseline platelet activity on platelet inhibition within 24hours post-STEMI. Methods and Findings 108 patients, treated with prasugrel and procedural bivalirudin, underwent Multiplate® platelet function testing at baseline, 0, 1, 2 and 24hours post-procedure. Major adverse cardiac events (MACE), bleeding and stent thrombosis (ST) were recorded. Baseline ADP activity was high (88.3U [71.8–109.0]), procedural time and consequently bivalirudin infusion duration were short (median door-to-end time 55minutes [40–70] and infusion duration 30minutes [20–42]). Baseline ADP was observed to influence all subsequent measurements of ADP activity, whereas door-to-end time only influenced ADP immediately post-procedure. High residual platelet reactivity (HRPR ADP>46.8U) was observed in 75% of patients immediately post-procedure and persisted in 24% of patients at 2hours. Five patients suffered in-hospital MACE (4.6%). Acute ST occurred in 4 patients, all were <120mins post-procedure and had HRPR. No significant bleeding was observed. In a post-hoc analysis, pre-procedural morphine use was associated with significantly higher ADP activity following intervention. Conclusions Baseline platelet function, time to STEMI treatment and opiate use all significantly influence immediate post-procedural platelet activity. PMID:26672598

  6. Mimicking adhesive functionalities of blood platelets using ligand-decorated liposomes.

    PubMed

    Ravikumar, Madhumitha; Modery, Christa L; Wong, Timothy L; Dzuricky, Michael; Sen Gupta, Anirban

    2012-06-20

    Platelet transfusion is used for treating a variety of bleeding complications. Natural platelet-based transfusion products have very short storage life (3-7 days) and high risks of biological contamination and side effects. Consequently, there is significant clinical interest in synthetic platelet-mimetic constructs that can promote hemostasis, while allowing convenient large-scale production, easy portability, long storage life, and minimal biological risks. To this end, research efforts are being directed toward particles that can amplify aggregation of activated platelets or can mimic platelet's ability to undergo adhesion to various vascular matrix proteins. Here, we report on a synthetic construct design that combines the mimicry of platelet's shear-dependent adhesion to vWF and shear-independent adhesion to collagen under flow, on a single particle. For this, we have used 150-nm-diameter liposomes as model particles and have decorated their surface simultaneously with vWF-binding and collagen-binding recombinant protein fragments or synthetic peptide motifs. We demonstrate in vitro that these surface-modified liposomes are able to adhere onto vWF surfaces in a shear-dependent fashion and onto collagen surfaces in a shear-independent fashion under flow. Moreover, when the vWF-binding and the collagen-binding were integrated on a single liposomal platform, the resultant heteromultivalent liposomes showed significantly enhanced adhesion to a vWF/collagen mixed surface compared to liposomes bearing vWF-binding or collagen-binding ligands only, as long as the ligand motifs did not spatially interfere with each other. Altogether, our results establish the feasibility of efficiently mimicking platelet's dual adhesion mechanisms on synthetic particles.

  7. Polymerization amplified SPR-DNA assay on noncovalently functionalized graphene.

    PubMed

    Yuan, Pei-Xin; Deng, Sheng-Yuan; Yao, Chuan-Guang; Wan, Ying; Cosnier, Serge; Shan, Dan

    2017-03-15

    A highly efficient surface plasmon resonance (SPR)-based DNA assay was developed, by employing noncovalently functionalized graphene nanosheets as a substrate, and enzymatic catalysis-induced polymerization as mass relay. The objective of this strategy was manifold: first of all, to sensitize the overall SPR output by in situ optimized electrogeneration of graphene thin-film, which was characterized by atomic force microscopic topography; secondly, to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid (NTA) scaffolds, that anchored onto graphene-supported pyrenyl derivatives; and lastly, to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters. With this setup, a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar, thus demonstrating a beneficial exploration and exploitation of two-dimensional nanomaterials as unique SPR infrastructure. The possibility of such ″bottom-up″ architecture mounted with ″top-down″ weight reactor would be most likely extensible and adaptable to protein determinations.

  8. 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

  9. 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.

  10. Megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained to survive and produce platelets

    PubMed Central

    Josefsson, Emma C.; James, Chloé; Henley, Katya J.; Debrincat, Marlyse A.; Rogers, Kelly L.; Dowling, Mark R.; White, Michael J.; Kruse, Elizabeth A.; Lane, Rachael M.; Ellis, Sarah; Nurden, Paquita; Mason, Kylie D.; O’Reilly, Lorraine A.; Roberts, Andrew W.; Metcalf, Donald; Huang, David C.S.

    2011-01-01

    It is believed that megakaryocytes undergo a specialized form of apoptosis to shed platelets. Conversely, a range of pathophysiological insults, including chemotherapy, are thought to cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. To resolve this paradox, we generated mice with hematopoietic- or megakaryocyte-specific deletions of the essential mediators of apoptosis, Bak and Bax. We found that platelet production was unperturbed. In stark contrast, deletion of the prosurvival protein Bcl-xL resulted in megakaryocyte apoptosis and a failure of platelet shedding. This could be rescued by deletion of Bak and Bax. We examined the effect on megakaryocytes of three agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and the BH3 mimetic ABT-737. All three triggered mitochondrial damage, caspase activation, and cell death. Deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and ABT-737. In vivo, mice with a Bak−/− Bax−/− hematopoietic system were protected against thrombocytopenia induced by the chemotherapeutic agent carboplatin. Thus, megakaryocytes do not activate the intrinsic pathway to generate platelets; rather, the opposite is true: they must restrain it to survive and progress safely through proplatelet formation and platelet shedding. PMID:21911424

  11. Essential Roles of Dopamine and Serotonin in Tooth Repair: Functional Interplay Between Odontogenic Stem Cells and Platelets.

    PubMed

    Baudry, Anne; Alleaume-Butaux, Aurélie; Dimitrova-Nakov, Sasha; Goldberg, Michel; Schneider, Benoît; Launay, Jean-Marie; Kellermann, Odile

    2015-08-01

    Characterizing stem cell intrinsic functions is an ongoing challenge for cell therapies. Here, we report that two independent A4 and H8 stem cell lines isolated from mouse molar pulp display the overall functions of bioaminergic cells. Both clones produce neurotrophins and synthesize, catabolize, store, and transport serotonin (5-hydroxytryptamine [5-HT]) and dopamine (DA). They express 5-HT1D,2B,7 and D1,3 autoreceptors, which render pulpal stem cells competent to respond to circulating 5-HT and DA. We show that injury-activated platelets are the source of systemic 5-HT and DA necessary for dental repair since natural dentin reparation is impaired in two rat models with monoamine storage-deficient blood platelets. Moreover, selective inhibition of either D1, D3, 5-HT2B, or 5-HT7 receptor within the pulp of wild-type rat molars after lesion alters the reparative process. Altogether our data argue that 5-HT and DA coreleased by pulp injury-activated platelets are critical for stem cell-mediated dental repair through 5-HT and DA receptor signalings.

  12. Targeting a Proteinase-Activated Receptor 4 (PAR4) Carboxyl Terminal Motif to Regulate Platelet Function.

    PubMed

    Ramachandran, Rithwik; Mihara, Koichiro; Thibeault, Pierre; Vanderboor, Christina M; Petri, Björn; Saifeddine, Mahmoud; Bouvier, Michel; Hollenberg, Morley D

    2017-04-01

    Thrombin initiates human platelet aggregation by coordinately activating proteinase-activated receptors (PARs) 1 and 4. However, targeting PAR1 with an orthosteric-tethered ligand binding-site antagonist results in bleeding, possibly owing to the important role of PAR1 activation on cells other than platelets. Because of its more restricted tissue expression profile, we have therefore turned to PAR4 as an antiplatelet target. We have identified an intracellular PAR4 C-terminal motif that regulates calcium signaling and β-arrestin interactions. By disrupting this PAR4 calcium/β-arrestin signaling process with a novel cell-penetrating peptide, we were able to inhibit both thrombin-triggered platelet aggregation in vitro and clot consolidation in vivo. We suggest that targeting PAR4 represents an attractive alternative to blocking PAR1 for antiplatelet therapy in humans.

  13. Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells

    PubMed Central

    2013-01-01

    Background Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. Results Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation. In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. Conclusions The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion. PMID:24168020

  14. The human megakaryocytic cell line UT-7/TPO expresses functional platelet agonist signals mediated through GPVI and thromboxane receptor.

    PubMed

    Kawaguchi, Tatsuya; Hashimoto, Ryuji; Yokota, Hiroshi

    2010-09-01

    We have demonstrated that a unique megakaryocytic cell line UT-7/TPO could respond to one of the primary platelet signals through GP (glycoprotein) VI and a secondary signal of the AA (arachidonic acid) cascade. Unlike other megakaryocytic cell lines, UT-7/TPO was found to express GPVI and its associate signal molecule of FcRgamma (Fc receptor gamma chain). When UT-7/TPO was stimulated with the GPVI agonist convulxin, the [Ca2+]i (intracellular Ca2+) was elevated in a convulxin concentration-dependent manner, and [Ca2+]i elevation was blocked by pretreatment with the Src family kinase inhibitor PP2 and the phospholipase inhibitor U73122. These results strongly indicate that endogenously expressed GPVI signal molecules are functional in UT-7/TPO. Concerning the AA cascade, the expression of COX (cyclooxygenase)-1 and TX (thromboxane) synthase was observed, and this cell line was able to produce TX by exogenous AA, followed by [Ca2+]i elevation mediated through the TX receptor. It is worth noting that convulxin stimulation did not cause TX generation, even through the GPVI pathway and the AA cascade are functional in this cell line. As there are many reports that convulxin-stimulated platelets failed to produce TX, it is suggested that UT-7/TPO has the same property as the platelets in regards to convulxin stimulation. Thus, UT-7/TPO is useful for the observation of both the GPVI pathway and AA cascade without requiring either the induction of differentiation or GPVI transfection. Furthermore, this cell line provides a new tool for research on platelet activation signals.

  15. Comparison of ticagrelor and high-dose clopidogrel on the platelet functions in patients with inadequate response to clopidogrel

    PubMed Central

    Gu, Xinshun; Fu, Xianghua; Wang, Yanbo; Zhang, Wenhui; Fan, Weize; Jiang, Yunfa; Hao, Guozhen; Miao, Qing; Li, Yi; Zhi, Wei

    2017-01-01

    Objective: To evaluate the effects of ticagrelor and high-dose clopidogrel on the platelet functions in patients with inadequate response to clopidogrel. Methods: In this prospective, randomized and controlled study, patients who had been diagnosed as acute coronary syndrome (ACS) with inadequate response to clopidogrel in the Second Hospital of Hebei Medical University from July 2015 to June 2016 were enrolled. Inadequate response to clopidogrel was defined as absolute reduction of platelet aggregation rate (PAR) <30% or PAR >70%. Eligible patients were randomly assigned to two groups, the high-dose group and the ticagrelor group. Clinical information and intervention protocols were compared. The PAR of the two groups were measured at the time of baseline, the 24th hour, 72nd hour, and the 7th day after treatments, the other platelet-related parameters were measured including platelet count (PLT), mean platelet volume (MPV), platelet distribution width (PDW) at the same time points. Besides, the markers of platelet activation human P-selectin (CD62P) and thromboxane A2 (TXA2) were also recorded to compare. The incidence of major adverse cardiac events (MACE) and the side effects between two groups were followed up for three months. Results: A total of 74 patients were finally enrolled, 38 of whom were assigned to the ticagrelor group and the rest of them to the high-dose clopidogrel group. The baseline clinical and procedural characterists were similar. There were no significant differences in baseline levels of PAR between the two groups [(79.38±11.20)% vs. (73.97±12.74)%, P>0.05]. For both groups, the levels of PAR significantly decreased at each time point (P<0.001). Besides, the levels of PAR in ticagrelor group were lower than those in high-dose clopidogrel group at the 24th hour, 72nd hour and 7th day after treatments: [(25.92±10.31)% vs. (37.95±11.63)%, P<0.001], [(28.02±14.61)% vs. (30.64±10.73)%, P<0.001], [(37.17±11.11)% vs. (36.80±7.26)%, P<0

  16. 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.

  17. Human endometrial stromal stem cells differentiate into megakaryocytes with the ability to produce functional platelets.

    PubMed

    Wang, Jinju; Chen, Shuzhen; Zhang, Cheng; Stegeman, Samantha; Pfaff-Amesse, Teresa; Zhang, Ying; Zhang, Wenfeng; Amesse, Lawrence; Chen, Yanfang

    2012-01-01

    Human endometrium is a high dynamic tissue that contains endometrial stromal stem cells (hESSCs). The hESSCs have been differentiated into a number of cell lineages. However, differentiation of hESSCs into megakaryocytes (MKs) has not yet been investigated. The aim of this study was to investigate the feasibility of MK generation from hESSCs and subsequent production of functional platelets (PLTs). In our study, hESSCs were cultured from endometrial stromal cells as confirmed by positive stromal cell specific markers (CD90 and CD29) and negative hematopoietic stem cell markers (CD45 and CD34) expression. Then, hESSCs were differentiated in a medium supplemented with thrombopoietin (TPO) for 18 days. The MK differentiation was analyzed by flow cytometry and confocal microscopy. The differentiation medium was collected for PLT production analysis by flow cytometry, transmission electron microscopy and functional measurements. Our results show: 1) MKs were successfully generated from hESSCs as identified by expression of specific markers (CD41a: 1 ± 0.09% and 39 ± 3.0%; CD42b: 1.2 ± 0.06% and 28 ± 2.0%, control vs. differentiation) accompanied with reduction of pluripotent transcription factors (Oct4 and Sox2) expression; 2) The level of PLTs in the differentiation medium was 16 ± 1 number/µl as determined by size (2-4 µm) and CD41a expression (CD41a: 1 ± 0.4% and 90±2.0%, control vs. differentiation); 3) Generated PLTs were functional as evidenced by the up-regulation of CD62p expression and fibrinogen binding following thrombin stimulation; 4) Released PLTs showed similar ultra-structure characteristics (alpha granules, vacuoles and dense tubular system) as PLTs from peripheral blood determined by electron microscopic analysis. Data demonstrate the feasibility of generating MKs from hESSCs, and that the generated MKs release functional PLTs. Therefore, hESSCs could be a potential new stem cell source for in vitro MK/PLT production.

  18. Beginner's luck--the first in vivo demonstration of functioning platelets; William Duke, 1910.

    PubMed

    Boulton, F

    2012-04-01

    Blood platelets remained obscure until the early 20th century although from the 1880 s claims that low numbers were associated with certain types of 'purpura' began to gain favour. This article re-appraises critically, but with due consideration to the limited technology of the times, the first remarkable in vivo demonstration of the effects of platelets demonstrated by the serial 'Bleeding Times' reported by William Duke in 1910, when fresh blood was transfused to two thrombocytopenic people. It also speculates on the possible causes of the thrombocytopenia with which Duke's main patient presented.

  19. 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.

  20. 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

  1. 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

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

    PubMed

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

    2015-05-01

    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 (2.6g/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.

  3. Platelet additive solution - electrolytes.

    PubMed

    Azuma, Hiroshi; Hirayama, Junichi; Akino, Mitsuaki; Ikeda, Hisami

    2011-06-01

    Recent attention to solutions that replace most or all plasma in platelet concentrates, while maintaining satisfactory platelet function, is motivated by the potential of plasma reduction or depletion to mitigate various transfusion-related adverse events. This report considers the electrolytic composition of previously described platelet additive solutions, in order to draw general conclusions about what is required for platelet function and longevity. The optimal concentrations of Na(+) and Cl(-) are 69-115 mM. The presence of both K(+) and Mg(2+) in platelet suspension at nearly physiological concentrations (3-5mM and 1.5-3mM, respectively) is indispensable for good preservation capacity because both electrolytes are required to prevent platelet activation. In contrast to K(+) and Mg(2+), Ca(2+) may not be important because no free Ca(2+) is available in M-sol, which showed excellent platelet preservation capacity at less than 5% plasma concentration. The importance of bicarbonate (approximately 40 mM) can be recognized when the platelets are suspended in additive solution under less than 5% residual plasma concentration.

  4. Evaluation of Dried Storage of Platelets for Transfusion: Physiologic Integrity and Hemostatic Functionality

    DTIC Science & Technology

    1994-10-27

    investigating a rat model. We have been successful in inducing thrombocytopenia in rats by treatment with an anti-rat thrombocyte antibody. With antibody...retraction that clots formed in the presence of rehydrated platelets undergo. We have used scanning electron microscopy (SEM) to examine clots formed in the

  5. Platelet Donation

    MedlinePlus

    ... donating platelets, can I still donate blood? What blood types should donate platelets? Can I donate plasma at ... Community Learn About Blood Blood Facts and Statistics Blood Types Blood Components What Happens to Donated Blood Blood ...

  6. 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.

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

    PubMed

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

    2016-11-01

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

  8. Edge-Selectively Functionalized Graphene-Like Platelets as a Co-curing Agent and a Nanoscale Additive to Epoxy Resin

    DTIC Science & Technology

    2012-08-12

    second method is where graphene can be grown using chemical vapor deposition (CVD) on a metal substrate18–20 or from single crystal silicon carbide...1 Final Report for AOARD Grant 114042 “Edge-selectively functionalized graphene -like platelets as a co-curing agent and a nanoscale additive...the role of a molecular wedge to exfoliate the AB-graphite into individual graphene and graphene -like platelets upon dispersion in polar solvents

  9. Early alterations in platelet mitochondrial function are associated with survival and organ failure in patients with septic shock

    PubMed Central

    Puskarich, Michael A.; Kline, Jeffrey A.; Watts, John A.; Shirey, Kristin; Hosler, Jonathan; Jones, Alan E.

    2015-01-01

    Introduction To determine if changes platelet mitochondrial function in patients with sepsis are present early following presentation, and the association of these changes with clinical outcomes and systemic metabolic function. Materials and Methods Prospective observational cohort study of a convenience sample of patients with severe sepsis. Mitochondrial function of intact, non-permeabilized platelets suspended in their own plasma was estimated using high resolution respirometry. Unstimulated basal respiration, oligomycin-induced state 4 (state 4o) and maximal respiratory rate following serial titrations of FCCP were measured. Organ failure was estimated using SOFA score, and patients were followed until 28 days to determine survival. Lactate levels were measured in all patients, and a subset of patients had lactate:pyruvate ratios measured. Results 28 patients were enrolled, 21 of whom survived. Initial SOFA score and lactate levels were 8.5 (IQR 6, 10) and 2.3 (IQR 1.2, 3.5) respectively, while the median L/P ratio was 23.4 (IQR 15.2, 38). Basal and maximal respiratory rates were significantly higher among non-survivors compared to survivors (p=0.02 and 0.04), while oligomycin-induced state 4 respiration was not statistically different between groups (p = 0.15). We found a significant association between maximal respiration and organ failure (p = 0.03), and both basal and maximal rates with initial lactate level (p = 0.04, 0.02), but not with lactate:pyruvate ratio. Conclusions Differences in platelet mitochondrial function between survivors and non-survivors are present very early in the hospital course and are associated with organ failure and lactate. PMID:26511963

  10. Dynamic platelet adhesion in patients with an acute coronary syndrome: The effect of antiplatelet therapy.

    PubMed

    Tsoumani, Maria E; Tatsidou, Prokopia T; Ntalas, Ioannis V; Goudevenos, John A; Tselepis, Alexandros D

    2016-12-01

    Platelet adhesion and aggregation are key functions leading to thrombus formation. The effect of aspirin, clopidogrel, and ticagrelor on platelet aggregation has been well established, however, there is limited data on the effect of these drugs on platelet adhesion. We therefore evaluated the effect of these drugs on platelet adhesion in acute coronary syndrome (ACS) patients. Citrated blood was collected from 50 ACS patients loaded with 325 mg of aspirin (baseline) and at 5 days after the administration of aspirin 100 mg/day and clopidogrel (600 mg loading dose, 75 mg/day) (n = 26) or ticagrelor (180 mg loading dose, 90 mg × 2/day) (n = 24). High on-treatment platelet reactivity (HTPR) to clopidogrel was estimated by vasodilator stimulated phosphoprotein (VASP) phosphorylation assay. Platelet adhesion to collagen was studied for 6 min under high shear stress and was evaluated using the time to platelet recruitment (TPR), the perimeter and average area of each adherent object, number of adherent objects, and the total percent of surface coverage (SC%). Six ACS patients exhibited HTPR to clopidogrel and excluded from the platelet adhesion assays. TPR and SC% values were similar among patient groups at baseline and controls. However, all other adhesion parameters were different in ACS patients, indicating the formation of more aggregates in regard to controls. At 5 days post-treatment with either clopidogrel or ticagrelor, the TPR values were increased and the SC% values were reduced to a similar extent compared with baseline. However, significant differences were observed in the ticagrelor group in the perimeter, number of adherent objects, and the average area of each adherent object indicating a more potent inhibition of adherence-induced platelet aggregation than clopidogrel. In conclusion, aspirin does not affect platelet adherence to collagen, whereas clopidogrel and ticagrelor inhibit to a similar extent dynamic platelet adhesion at 5 days post-treatment in

  11. Application of an optimized flow cytometry-based quantification of Platelet Activation (PACT): Monitoring platelet activation in platelet concentrates

    PubMed Central

    Roest, Mark; Henskens, Yvonne M. C.; de Laat, Bas; Huskens, Dana

    2017-01-01

    Background Previous studies have shown that flow cytometry is a reliable test to quantify platelet function in stored platelet concentrates (PC). It is thought that flow cytometry is laborious and hence expensive. We have optimized the flow cytometry-based quantification of agonist induced platelet activation (PACT) to a labor, time and more cost-efficient test. Currently the quality of PCs is only monitored by visual inspection, because available assays are unreliable or too laborious for use in a clinical transfusion laboratory. Therefore, the PACT was applied to monitor PC activation during storage. Study design and methods The optimized PACT was used to monitor 5 PCs during 10 days of storage. In brief, optimized PACT uses a ready-to-use reaction mix, which is stable at -20°C. When needed, a test strip is thawed and platelet activation is initiated by mixing PC with PACT. PACT was based on the following agonists: adenosine diphosphate (ADP), collagen-related peptide (CRP) and thrombin receptor-activating peptide (TRAP-6). Platelet activation was measured as P-selectin expression. Light transmission aggregometry (LTA) was performed as a reference. Results Both PACT and LTA showed platelet function decline during 10-day storage after stimulation with ADP and collagen/CRP; furthermore, PACT showed decreasing TRAP-induced activation. Major differences between the two tests are that PACT is able to measure the status of platelets in the absence of agonists, and it can differentiate between the number of activated platelets and the amount of activation, whereas LTA only measures aggregation in response to an agonist. Also, PACT is more time-efficient compared to LTA and allows high-throughput analysis. Conclusion PACT is an optimized platelet function test that can be used to monitor the activation of PCs. PACT has the same accuracy as LTA with regard to monitoring PCs, but it is superior to both LTA and conventional flow cytometry based tests with regard to labor

  12. 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

  13. [Importance of the interaction of surface receptors with the cytoskeleton for platelet function].

    PubMed

    Meyer, M

    1990-01-01

    Two Triton-X-100-insoluble cytoskeletal fractions were prepared from washed human blood platelets by centrifugation: a low speed sediment (TP) and a fraction recovered as ultracentrifugation pellet (UP). Glycoproteins associated with both fractions prior to and after different times of thrombin activation were analyzed by electrophoretic techniques. In the TP fraction of unstimulated platelet the major membrane glycoproteins (GP) Ib and Ia were detected. After thrombin stimulation there is a rapid increase of GPs Ia, Ib, IIIb and a Mr 250,000-GP and a slower appearance of GPs IIb and IIIa in this fraction. The UP fraction contains mainly GPs Ib, Ia, IIb, IIIa and a Mr 250,000-GP. Thrombin activation does not cause any significant changes in the glycopeptide pattern of this fraction.

  14. 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.

  15. A Functional High-Throughput Assay of Myelination in Vitro

    DTIC Science & Technology

    2014-07-01

    Human induced pluripotent stem cells , hydrogels, 3D culture, electrophysiology, high-throughput assay 16. SECURITY CLASSIFICATION OF: 17...clear that four of the seven human astrocyte cell lines (HA #1, 2, 3, and 7) show very large amounts of neuronal differentiation when using epigenetic...derived.   5    Fig. 1: Spontaneous differentiation toward neuronal lineage of iPS cells derived from human astrocytes. Left: phase contrast

  16. Platelet Adhesion under Flow

    PubMed Central

    Ruggeri, Zaverio M.

    2011-01-01

    Platelet adhesive mechanisms play a well-defined role in hemostasis and thrombosis, but evidence continues to emerge for a relevant contribution to other pathophysiological processes including inflammation, immune-mediated responses to microbial and viral pathogens, and cancer metastasis. Hemostasis and thrombosis are related aspects of the response to vascular injury, but the former protects from bleeding after trauma while the latter is a disease mechanism. In either situation, adhesive interactions mediated by specific membrane receptors support the initial attachment of single platelets to cellular and extracellular matrix constituents of the vessel wall and tissues. In the subsequent steps of thrombus growth and stabilization, adhesive interactions mediate platelet to platelet cohesion (aggregation) and anchoring to the fibrin clot. A key functional aspect of platelets is their ability to circulate in a quiescent state surveying the integrity of the inner vascular surface, coupled to a prompt reaction wherever alterations are detected. In many respects, therefore, platelet adhesion to vascular wall structures, to one another or to other blood cells are facets of the same fundamental biological process. The adaptation of platelet adhesive functions to the effects of blood flow is the main focus of this review. PMID:19191170

  17. Physiologic and pathologic changes of platelets in pregnancy.

    PubMed

    Valera, Marie-Cecile; Parant, Olivier; Vayssiere, Christophe; Arnal, Jean-François; Payrastre, Bernard

    2010-01-01

    Platelets are key players in haemostasis and thrombus formation. Defects affecting platelets during pregnancy can lead to heterogeneous complications, such as thrombosis, first trimester miscarriage and postpartum haemorrhage. The incidence of complications is increased in women who have heritable platelet function disorders. Modifications of platelet count or platelet functions during normal pregnancy and preeclampsia will be summarized and the management of pregnant women with heritable platelet function disorders will be discussed.

  18. Staphylococcal β-Toxin Modulates Human Aortic Endothelial Cell and Platelet Function through Sphingomyelinase and Biofilm Ligase Activities

    PubMed Central

    Herrera, Alfa; Kulhankova, Katarina; Sonkar, Vijay K.; Dayal, Sanjana; Klingelhutz, Aloysius J.; Salgado-Pabón, Wilmara

    2017-01-01

    ABSTRACT Staphylococcus aureus causes many infections, such as skin and soft tissue, pneumonia, osteomyelitis, and infective endocarditis (IE). IE is an endovascular infection of native and prosthetic valves and the lining of the heart; it is characterized by the formation of cauliflower-like “vegetations” composed of fibrin, platelets, other host factors, bacteria, and bacterial products. β-Toxin is an S. aureus virulence factor that contributes to the microorganism’s ability to cause IE. This cytolysin has two enzymatic activities: sphingomyelinase (SMase) and biofilm ligase. Although both activities have functions in a rabbit model of IE, the mechanism(s) by which β-toxin directly affects human cells and is involved in the infectious process has not been elucidated. Here, we compared the in vitro effects of purified recombinant wild-type β-toxin, SMase-deficient β-toxin (H289N), and biofilm ligase-deficient β-toxin (H162A and/or D163A) on human aortic endothelial cells (HAECs) and platelets. β-Toxin was cytotoxic to HAECs and inhibited the production of interleukin 8 (IL-8) from these cells by both SMase and biofilm ligase activities. β-Toxin altered HAEC surface expression of CD40 and vascular cell adhesion molecule 1 (VCAM-1). HAECs treated with β-toxin displayed granular membrane morphology not seen in treatment with the SMase-deficient mutant. The altered morphology resulted in two possibly separable activities, cell rounding and redistribution of cell membranes into granules, which were not the result of endosome production from the Golgi apparatus or lysosomes. β-Toxin directly aggregated rabbit platelets via SMase activity. PMID:28325766

  19. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    DOE PAGES

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Westernmore » blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.« less

  20. 5-Hydroxytryptamine-2A receptor gene (HTR 2 A) candidate polymorphism (T 102 C): Role for human platelet function under pharmacological challenge ex vivo.

    PubMed

    Ozdener, F; Gülbas, Z; Erol, K; Ozdemir, V

    2005-01-01

    Although the environmental and life-style factors influencing individual predisposition to acute myocardial infarction (AMI) have been well documented, little is known on the identity of genetic loci that may contribute to risk for AMI. Recently, genetic studies in patients with nonfatal AMI have suggested an association with the T 102 C polymorphism in the serotonin 5-HT(2A) receptor gene (HTR 2 A). Considering the significant role of the 5-HT(2A) receptor in serotonin-induced platelet responses and the contribution of platelet (patho)physiology to thromboembolic events, we postulated that the increased susceptibility to AMI in patients with the T 102 homozygosity may be attributable, in part, to altered serotonin-mediated platelet function. In a group of healthy volunteers recruited from the Eskisehir region in central Turkey (N=37), we investigated the functional consequences of HTR 2 A T 102 C polymorphism in relation to platelet pharmacodynamics ex vivo. The platelet shape change and aggregation response to serotonin were measured with use of the platelet aggregometry and expressed as aggregometer output (mm). Because the circulating catecholamine hormone epinephrine can augment platelet aggregation, pharmacodynamic response (aggregation and its inhibition by 5-HT(2A) receptor antagonist cyproheptadine) was measured in the presence of both serotonin and epinephrine, to mimic the clinical situation in patients. The mean platelet aggregation was higher by 38% in subjects with T 10 2 homozygosity (T/T genotype, N=13) when compared with the carriers of the 102 C-allele (T/C and the C/C genotypic groups, N=24) (39.5 mm+/-12.3 vs. 28.7 mm+/-16.8, respectively) (mean+/-SD) (p<0.05). On the other hand, neither the serotonin-induced platelet shape change nor the cyproheptadine inhibition of platelet aggregation was influenced by the HTR 2 A T 102 C genetic variation (p>0.05). These findings in healthy subjects may provide a mechanistic explanation for the previously

  1. Assessing blood-brain barrier function using in vitro assays.

    PubMed

    Bressler, Joseph; Clark, Katherine; O'Driscoll, Cliona

    2013-01-01

    The impermeability of the blood-brain barrier (BBB) is due to a number of properties including tight junctions on adjoining endothelial cells, absence of pinocytic vesicles, and expression of multidrug transporters. Although the permeability of many chemicals can be predicted by their polarity, or oil/water partition coefficient, many lipophilic chemicals are not permeable because of multidrug transporters at the luminal and abluminal membranes. In contrast, many nutrients, which are usually polar, cross the BBB more readily than predicted by their oil/water partition coefficients due to the expression of specific nutrient transporters. In vitro models are being developed because rodent models are of low input and relatively expensive. Isolated brain microvessels and cell culture models each offers certain advantages and disadvantages. Isolated brain microvessels are useful in measuring multidrug drug transporters and tight junction integrity, whereas cell culture models allow the investigator to measure directional transport and can be genetically manipulated. In this chapter, we describe how to isolate large batches of brain microvessels from freshly slaughtered cows. The different steps in the isolation procedure include density gradient centrifugations and filtering. Purity is determined microscopically and by marker enzymes. Permeability is assessed by measuring the uptake of fluorescein-labeled dextran in an assay that has been optimized to have a large dynamic range and low inter-day variability. We also describe how to evaluate transendothelial cell electrical resistance and paracellular transport in cell culture models.

  2. An assay for functional xylose transporters in Saccharomyces cerevisiae.

    PubMed

    Wang, Chengqiang; Shen, Yu; Hou, Jin; Suo, Fan; Bao, Xiaoming

    2013-11-15

    It has been considered that more efficient uptake of xylose could promote increased xylose metabolic capacity of several microorganisms. In this study, an assay to screen xylose transporters was established in the Saccharomyces cerevisiae strain, which expresses the xylosidase gene of Bacillus pumilus intracellularly. The absorbed xylose analog p-nitrophenyl-β-d-xylopyranoside (pNPX) rapidly hydrolyzed to p-nitrophenol (pNP), which displayed a yellow tint when exposed to xylosidase in vivo. The xylose transporter activities of the strain were computed using the pNP production rate, which was detected extracellularly. This method could be used for both high-throughput screening and smaller scale investigations. AraEp, which is a pentose transporter of Corynebacterium glutamicum, was expressed in S. cerevisiae and exhibited better transport capacity than the endogenous transporters Hxt7p and Gal2p. Moreover, a mutant of AraEp with 103% greater transport capacity was screened out, and the computer simulation suggested that transmembrane domain 5 was an important factor for the transport capacity of AraEp in S. cerevisiae.

  3. Using transgenic reporter assays to functionally characterize enhancers in animals.

    PubMed

    Kvon, Evgeny Z

    2015-09-01

    Enhancers or cis-regulatory modules play an instructive role in regulating gene expression during animal development and in response to the environment. Despite their importance, we only have an incomplete map of enhancers in the genome and our understanding of the mechanisms governing their function is still limited. Recent advances in genomics provided powerful tools to generate genome-wide maps of potential enhancers. However, most of these methods are based on indirect measures of enhancer activity and have to be followed by functional testing. Animal transgenesis has been a valuable method to functionally test and characterize enhancers in vivo. In this review I discuss how different transgenic strategies are utilized to characterize enhancers in model organisms focusing on studies in Drosophila and mouse. I will further discuss recent large-scale transgenic efforts to systematically identify and catalog enhancers as well as highlight the challenges and future directions in the field.

  4. Microfluidic whole blood testing of platelet response to pharmacological agents.

    PubMed

    Li, Ruizhi; Grosser, Tilo; Diamond, Scott L

    2017-01-19

    Platelets present a number of intracellular and transmembrane targets subject to pharmacological modulation, either for cardiovascular disease reduction or as an unintended drug response. Microfluidic devices allow human blood to clot on a defined surface under controlled hemodynamic and pharmacological conditions. The potencies of a number of antiplatelet and anticancer drugs have been tested with respect to platelet deposition on collagen under flow. Inhibitors of cyclooxygenase-1 (COX-1) reduce platelet deposition, either when added ex vivo to blood or ingested orally by patients prior to testing. Some individuals display a functional "aspirin-insensitivity" in microfluidic assay. When certain nonsteroidal anti-inflammatory drugs (NSAIDs) are taken orally, they block COX-1 acetylation by aspirin with concomitant reduction of aspirin efficacy against platelets in microfluidic assay. Both P2Y1 and P2Y12 inhibitors reduce platelet deposition under flow, as do NO donors and iloprost that target the guanylate cyclase and the prostacyclin receptor, respectively. In a microfluidic assay of 37 kinase inhibitors, dasatinib had potent antiplatelet activity, while bosutinib was less potent. Dasatinib and bosutinib have known profiles against numerous kinases, revealing overlapping and nonoverlapping activities relevant to their unique actions against platelets. Also, dasatinib caused a marked and specific inhibition of GPVI signaling induced by convulxin, consistent with a dasatinib-associated bleeding risk. Microfluidic devices facilitate drug library screening, dose-response testing, and drug-drug interaction studies. Kinase inhibitors developed as anticancer agents may present antiplatelet activities that are detectable by microfluidic assay and potentially linked to bleeding risks.

  5. 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.

  6. The role of RNA uptake in platelet heterogeneity.

    PubMed

    Clancy, Lauren; Beaulieu, Lea M; Tanriverdi, Kahraman; Freedman, Jane E

    2017-03-09

    The role of platelets in regulating vascular homeostasis has expanded beyond mediation of haemostasis and thrombosis. The discovery of platelet RNA and the presence of subpopulations of platelets containing varying amounts of RNA suggest a role for platelet transcripts in vascular function. As the RNA in anucleated platelets is biologically functional and may transfer to other vascular cells, we hypothesised that platelet RNA diminishes over the lifespan of the platelet with diminishing platelet size due to horizontal cellular transfer. The purpose of this study is to determine if platelet RNA variance is the result of horizontal cellular transfer between platelets and other vascular cells. Utilising platelet sorting and RNA sequencing, we found that smaller platelets contained a more diverse set of transcripts than larger platelets. Further investigation using fluorescence imaging, gene expression analyses and in vitro and in vivo modelling revealed that platelets take up RNA from other vascular cells in a complex manner, revealing a dynamic role for platelets in modulating vascular homeostasis through bidirectional RNA transfer. The resultant RNA profile heterogeneity suggests unique functional roles for platelets dependent on size and complexity. This study expands our basic understanding of platelet function and heterogeneity and is the first to evaluate endogenous vascular RNA uptake and its relation to platelet processes. Our findings describe a novel endogenous phenomenon that can help elucidate the platelet's role in these non-thrombotic and haemostatic fields, as well as present potential for diagnostic and therapeutic development.

  7. T granules in human platelets function in TLR9 organization and signaling.

    PubMed

    Thon, Jonathan N; Peters, Christopher G; Machlus, Kellie R; Aslam, Rukhsana; Rowley, Jesse; Macleod, Hannah; Devine, Matthew T; Fuchs, Tobias A; Weyrich, Andrew S; Semple, John W; Flaumenhaft, Robert; Italiano, Joseph E

    2012-08-20

    Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs.

  8. Yeast-based reporter assays for the functional characterization of cochaperone interactions with steroid hormone receptors.

    PubMed

    Balsiger, Heather A; Cox, Marc B

    2009-01-01

    Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter describes a hormone-inducible androgen receptor-mediated beta-galactosidase reporter assay in yeast. In addition, the immunophilin FKBP52 is used as a specific example of a receptor-associated cochaperone that acts as a positive regulator of receptor function. With the right combination of receptor and cochaperone expression plasmids, reporter plasmid, and ligand, the assay protocol described here could be used to functionally characterize a wide variety of nuclear receptor-cochaperone interactions. In addition to the functional characterization of receptor regulatory proteins, a modified version of this assay is currently being used to screen compound libraries for selective FKBP52 inhibitors that represent attractive therapeutic candidates for the treatment of steroid hormone receptor-associated diseases.

  9. Autologous platelet-labeling in thrombocytopenia

    SciTech Connect

    Sinzinger, H.; Virgolini, I.; Vinazzer, H. )

    1990-11-01

    Field studies performed with peripheral platelets obtained from 6 male volunteers aged 23 to 29 years revealed an extraordinary dependence of labeling efficiency on incubation time and platelet concentration after {sup 111}In-oxine platelet labeling. Since the monitoring of in vivo-platelet function in patients with thrombocytopenia may cause problems due to insufficient labeling results and homologous platelets may show a different in vivo behaviour to autologous ones, we have searched for the minimal amount of platelets necessary to allow appropriate labeling and imaging in patients with thrombocytopenia. In 15 patients with untreated thrombocytopenia aged 14 to 79 years demonstrating a mean peripheral platelet count of 2.509 +/- 1.45 x 10(4) cells/microliters autologous {sup 111}In-oxine platelet labeling was performed. The results indicate that approximately 1 x 10(8) (concentrated) platelets/ml are necessary to obtain an adequate labeling efficiency and recovery. This platelet concentration can be easily achieved by drawing one more Monovette of whole blood per each 5 x 10(4) platelets/microliter peripheral platelet count less than 2 x 10(5)/microliter. It is concluded, that calculation of the required number of platelets in advance, variation of the blood volume drawn and the volume of incubation buffer allow informative, qualitative and quantitative results using autologous platelets. The method presented effectively circumvents the requirement of homologous platelets for radiolabeling in thrombocytopenia.

  10. [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.

  11. Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis.

    PubMed

    Slungaard, A; Key, N S

    1994-10-14

    Thrombomodulin (TM) is an anionic (pI approximately 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED50 of 3.3-10 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km for protein C of APC generation by GAG+ TM. PF4 also strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in native (but not Gla-domainless) protein C conformation as assessed by autofluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.

  12. Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

    PubMed

    Lieb, S; Littmann, T; Plank, N; Felixberger, J; Tanaka, M; Schäfer, T; Krief, S; Elz, S; Friedland, K; Bernhardt, G; Wegener, J; Ozawa, T; Buschauer, A

    2016-12-01

    A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

  13. 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.

  14. Patterning surfaces for controlled platelet adhesion and detection of dysfunctional platelets.

    PubMed

    Ye, Wei; Shi, Qiang; Wong, Shing-Chung; Hou, Jianwen; Shi, Hengchong; Yin, Jinghua

    2013-06-01

    Platelets play a fundamental role in thrombus formation and in the pathogenesis of arterial thrombosis. Patterning surfaces for controlled platelet adhesion paves the way for adhesion and activation mechanisms in platelets and detection of platelet functional defects. Here, a new and simple method based on controlled polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) on the surface of styrene-block-(ethylene-co-butylene)-block-styrene (SEBS) is shown. The competition between polymerization and degradation enables platelet adhesion on SEBS to be switched on and off. The adhesive sites of the platelets can be down to single cell level, and the dysfunctional platelets can be quantitatively detected.

  15. A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time.

    PubMed

    Six, Katrijn R; Devloo, Rosalie; Van Aelst, Britt; Vandekerckhove, Philippe; Feys, Hendrik B; Compernolle, Veerle

    2017-02-14

    Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca(2+)-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca(2+)/Mg(2+) buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca(2+) and Mg(2+) levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

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

    NASA Astrophysics Data System (ADS)

    Li, Dongdong; Zhang, Yuping; Zhou, Bingbing

    2015-05-01

    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.

  17. Recent advances in functional assays of transcriptional enhancers.

    PubMed

    Babbitt, Courtney C; Markstein, Michele; Gray, Jesse M

    2015-09-01

    In this special edition of Genomics, we present reviews of the current state of the field in identifying and functionally understanding transcriptional enhancers in cells and developing tissues. Typically several enhancers coordinate the expression of an individual target gene, each controlling that gene's expression in specific cell types at specific times. Until recently, identifying each gene's enhancers had been challenging because enhancers do not occupy prescribed locations relative to their target genes. Recently there have been powerful advances in DNA sequencing and other technologies that make it possible to identify the majority of enhancers in virtually any cell type of interest. The reviews in this edition of Genomics highlight some of these new and powerful approaches.

  18. Future innovations in anti-platelet therapies

    PubMed Central

    Barrett, N E; Holbrook, L; Jones, S; Kaiser, W J; Moraes, L A; Rana, R; Sage, T; Stanley, R G; Tucker, K L; Wright, B; Gibbins, J M

    2008-01-01

    Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed. PMID:18587441

  19. A rapid membrane potential assay to monitor CFTR function and inhibition.

    PubMed

    Maitra, Rangan; Sivashanmugam, Perumal; Warner, Keith

    2013-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an important regulator of ion transport and fluid secretion in humans. Mutations to CFTR cause cystic fibrosis, which is a common recessive genetic disorder in Caucasians. Involvement of CFTR has been noted in other important diseases, such as secretory diarrhea and polycystic kidney disease. The assays to monitor CFTR function that have been described to date either are complicated or require specialized instrumentation and training for execution. In this report, we describe a rapid FlexStation-based membrane potential assay to monitor CFTR function. In this assay, agonist-mediated activation of CFTR results in membrane depolarization that can be monitored using a fluorescent membrane potential probe. Availability of a simple mix-and-read assay to monitor the function of this important protein might accelerate the discovery of CFTR ligands to study a variety of conditions.

  20. Platelets and diabetes mellitus.

    PubMed

    Santilli, Francesca; Simeone, Paola; Liani, Rossella; Davì, Giovanni

    2015-07-01

    Platelet activation plays a key role in atherothrombosis in type 2 diabetes mellitus (T2DM) and increased in vivo platelet activation with enhanced thromboxane (TX) biosynthesis has been reported in patients with impairment of glucose metabolism even in the earlier stages of disease and in the preclinical phases. In this regards, platelets appear as addresses and players carrying and transducing metabolic derangement into vascular injury. The present review critically addresses key pathophysiological aspects including (i) hyperglycemia, glycemic variability and insulin resistance as determinants and predictors of platelet activation, (ii) inflammatory mediators derived from platelets, such as soluble CD40 ligand, soluble CD36, Dickkopf-1 and probably soluble receptor for advanced glycation-end-products (sRAGE), which expand the functional repertoire of platelets from players of hemostasis and thrombosis to powerful amplifiers of inflammation by promoting the release of cytokines and chemokines, cell activation, and cell-cell interactions; (iii) molecular mechanisms underpinning the less-than-expected antithrombotic protection by aspirin (ASA), despite regular antiplatelet prophylaxis at the standard dosing regimen, and (iv) stratification of patients deserving different antiplatelet strategies, based on the metabolic phenotype. Taken together, these pathophysiological aspects may contribute to the development of promising mechanism-based therapeutic strategies to reduce the progression of atherothrombosis in diabetic subjects.

  1. Homologous recombination in Sulfolobus acidocaldarius: genetic assays and functional properties.

    PubMed

    Grogan, Dennis W

    2009-02-01

    HR (homologous recombination) is expected to play important roles in the molecular biology and genetics of archaea, but, so far, few functional properties of archaeal HR have been measured in vivo. In the extreme thermoacidophile Sulfolobus acidocaldarius, a conjugational mechanism of DNA transfer enables quantitative analysis of HR between chromosomal markers. Early studies of this system indicated that HR occurred frequently between closely spaced mutations within the pyrE gene, and this result was later supported by various analyses involving defined point mutations and deletions. These properties of intragenic HR suggested a non-reciprocal mechanism in which donor sequences become incorporated into the recipient genome as short segments. Because fragmentation of donor DNA during cell-to-cell transfer could not be excluded from contributing to this result, subsequent analyses have focused on electroporation of selectable donor DNA directly into recipient strains. For example, S. acidocaldarius was found to incorporate synthetic ssDNA (single-stranded DNA) of more than approximately 20 nt readily into its genome. With respect to various molecular properties of the ssDNA substrates, the process resembled bacteriophage lambdaRed-mediated 'recombineering' in Escherichia coli. Another approach used electroporation of a multiply marked pyrE gene to measure donor sequence tracts transferred to the recipient genome in individual recombination events. Initial results indicate multiple discontinuous tracts in the majority of recombinants, representing a relatively broad distribution of tract lengths. This pattern suggests that properties of the HR process could, in principle, account for many of the apparent peculiarities of intragenic recombination initiated by S. acidocaldarius conjugation.

  2. Novel approaches for diagnosing inherited platelet disorders.

    PubMed

    Bastida Bermejo, José María; Hernández-Rivas, Jesús María; González-Porras, José Ramón

    2017-01-20

    Inherited platelet disorders diagnosis is based on the clinical history and bleeding assessment tools. The laboratory functional assays as well as the molecular test to identify the pathogenic genetic variant are essential to confirm the accurate diagnosis of these disorders. Nowadays, the main challenges to developing a new diagnostic system are involved in reducing the samples' volume, and faster and more helpful analysis. Moreover, there are no widely available and standardised global tests. High throughput genetic testing such as next-generation sequencing has revolutionised DNA sequencing technologies as it allows the simultaneous and faster investigation of multiple genes at a manageable cost. This technology has improved the molecular characterisation of inherited platelet disorders and has been implemented in the research studies and the clinical routine practice.

  3. 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

  4. Platelets can enhance vascular permeability.

    PubMed

    Cloutier, Nathalie; Paré, Alexandre; Farndale, Richard W; Schumacher, H Ralph; Nigrovic, Peter A; Lacroix, Steve; Boilard, Eric

    2012-08-09

    Platelets survey blood vessels, searching for endothelial damage and preventing loss of vascular integrity. However, there are circumstances where vascular permeability increases, suggesting that platelets sometimes fail to fulfill their expected function. Human inflammatory arthritis is associated with tissue edema attributed to enhanced permeability of the synovial microvasculature. Murine studies have suggested that such vascular leak facilitates entry of autoantibodies and may thereby promote joint inflammation. Whereas platelets typically help to promote microvascular integrity, we examined the role of platelets in synovial vascular permeability in murine experimental arthritis. Using an in vivo model of autoimmune arthritis, we confirmed the presence of endothelial gaps in inflamed synovium. Surprisingly, permeability in the inflamed joints was abrogated if the platelets were absent. This effect was mediated by platelet serotonin accumulated via the serotonin transporter and could be antagonized using serotonin-specific reuptake inhibitor antidepressants. As opposed to the conventional role of platelets to microvascular leakage, this demonstration that platelets are capable of amplifying and maintaining permeability adds to the rapidly growing list of unexpected functions for platelets.

  5. 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

  6. Platelet hyaluronidase-2: an enzyme that translocates to the surface upon activation to function in extracellular matrix degradation

    PubMed Central

    Albeiroti, Sami; Ayasoufi, Katayoun; Hill, David R.; Shen, Bo

    2015-01-01

    Following injury, platelets rapidly interact with the exposed extracellular matrix (ECM) of the vessel wall and the surrounding tissues. Hyaluronan (HA) is a major glycosaminoglycan component of the ECM and plays a significant role in regulating inflammation. We have recently reported that human platelets degrade HA from the surfaces of activated endothelial cells into fragments capable of inducing immune responses by monocytes. We also showed that human platelets contain the enzyme hyaluronidase-2 (HYAL2), one of two major hyaluronidases that digest HA in somatic tissues. The deposition of HA increases in inflamed tissues in several inflammatory diseases, including inflammatory bowel disease (IBD). We therefore wanted to define the mechanism by which platelets degrade HA in the inflamed tissues. In this study, we show that human platelets degrade the proinflammatory matrix HA through the activity of HYAL2 and that platelet activation causes the immediate translocation of HYAL2 from a distinct population of α-granules to platelet surfaces where it exerts its catalytic activity. Finally, we show that patients with IBD have lower platelet HYAL2 levels and activity than healthy controls. PMID:25411425

  7. Platelet hyaluronidase-2: an enzyme that translocates to the surface upon activation to function in extracellular matrix degradation.

    PubMed

    Albeiroti, Sami; Ayasoufi, Katayoun; Hill, David R; Shen, Bo; de la Motte, Carol A

    2015-02-26

    Following injury, platelets rapidly interact with the exposed extracellular matrix (ECM) of the vessel wall and the surrounding tissues. Hyaluronan (HA) is a major glycosaminoglycan component of the ECM and plays a significant role in regulating inflammation. We have recently reported that human platelets degrade HA from the surfaces of activated endothelial cells into fragments capable of inducing immune responses by monocytes. We also showed that human platelets contain the enzyme hyaluronidase-2 (HYAL2), one of two major hyaluronidases that digest HA in somatic tissues. The deposition of HA increases in inflamed tissues in several inflammatory diseases, including inflammatory bowel disease (IBD). We therefore wanted to define the mechanism by which platelets degrade HA in the inflamed tissues. In this study, we show that human platelets degrade the proinflammatory matrix HA through the activity of HYAL2 and that platelet activation causes the immediate translocation of HYAL2 from a distinct population of α-granules to platelet surfaces where it exerts its catalytic activity. Finally, we show that patients with IBD have lower platelet HYAL2 levels and activity than healthy controls.

  8. SPR-based assays enable the full functional analysis of bispecific molecules.

    PubMed

    Meschendoerfer, W; Gassner, C; Lipsmeier, F; Regula, J T; Moelleken, J

    2017-01-05

    The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins raises new challenges for functional characterization. When compared to standard antibodies, two individual interactions and the inter-dependency of binding events need to be considered for bispecific antibodies. We have previously described an SPR-based assay setup, which enables us to assess the binding activity of a bivalent-bispecific molecule to both targets simultaneously and - in addition to one individual target - in a single setup. However, there might be some pitfalls when applying the bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an alternative SPR-based assay principle, which allows the individual assessment of both targets in solution. Comparison of data between the assays showed that simultaneous binding can be calculated based on both individual readouts, and revealed a good correlation. Hence, both SPR-based assay principles allow a "full" functional analysis of a bispecific CrossMab in only one assay. The assay principles can be qualified and enable an efficient drug development.

  9. A point mutation in the EGF-4 domain of β(3) integrin is responsible for the formation of the Sec(a) platelet alloantigen and affects receptor function.

    PubMed

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

    2012-01-01

    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 (Sec(a)) residing on αIIbβ3. The location of Sec(a) on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the β3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbβ3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) 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 Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn 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 Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbβ3 receptor function.

  10. 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.

  11. Maresin-like lipid mediators are produced by leukocytes and platelets and rescue reparative function of diabetes-impaired macrophages.

    PubMed

    Hong, Song; Lu, Yan; Tian, Haibin; Alapure, Bhagwat V; Wang, Quansheng; Bunnell, Bruce A; Laborde, James Monroe

    2014-10-23

    Nonhealing diabetic wounds are associated with impaired macrophage (Mf) function. Leukocytes and platelets (PLT) play crucial roles in wound healing by poorly understood mechanisms. Here we report the identification and characterization of the maresin-like(L) mediators 14,22-dihydroxy-docosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acids, 14S,22-diHDHA (maresin-L1), and 14R,22-diHDHA (maresin-L2) that are produced by leukocytes and PLT and involved in wound healing. We show that 12-lipoxygenase-initiated 14S-hydroxylation or cytochrome P450 catalyzed 14R-hydroxylation and P450-initiated ω(22)-hydroxylation are required for maresin-L biosynthesis. Maresin-L treatment restores reparative functions of diabetic Mfs, suggesting that maresin-Ls act as autocrine/paracrine factors responsible for, at least in part, the reparative functions of leukocytes and PLT in wounds. Additionally, maresin-L ameliorates Mf inflammatory activation and has the potential to suppress the chronic inflammation in diabetic wounds caused by activation of Mfs. These findings provide initial insights into maresin-L biosynthesis and mechanism of action and potentially offer a therapeutic option for better treatment of diabetic wounds.

  12. 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.

  13. 2B or not 2B? Disparate discrimination of functional VWF discordance using different assay panels or methodologies may lead to success or failure in the early identification of type 2B VWD.

    PubMed

    Favaloro, Emmanuel J; Bonar, Roslyn; Meiring, Muriel; Street, Alison; Marsden, Katherine

    2007-08-01

    Laboratory proficiency in the identification of functional von Willebrand factor (VWF) discordance in type 2B von Willebrand disease (VWD) was assessed by external quality assurance surveys conducted by the RCPA Haematology QAP, and using six different type 2B VWD plasma samples (three historical and three previously unpublished) tested by up to 52 laboratories. For the three most recent samples, functional VWF discordance was either not identified in testing or by interpretation with misidentification as 'normal' or 'type 1 VWD', on average for 25.7% of test occasions when laboratories performed VWF:Ag and VWF:RCo as their primary VWF test panel, but somewhat fewer occasions (10.9%) for laboratories that incorporated VWF:CB as an additional functional VWF assay. VWF assay sub-methodologies also influenced the appropriate identification of samples as potentially type 2 VWD, and VWF functional discordance was more consistently identified when laboratories used (i) automated platelet agglutination for VWF:RCo compared to classical platelet aggregometry, (ii) inhouse VWF:CB assays compared to commercial kit methods, and (iii) automated LIA-based 'VWF:Activity' assays compared to ELISA based assays. We conclude that:(i) laboratories are generally proficient in tests for VWD but interpretative diagnostic errors do occur; (ii) correct diagnosis is more likely when test panels are more comprehensive and include the VWF:CB; (iii) sub-methodology influences the appropriate identification of VWF functional discordance. On the basis of these findings, we provide a series of recommendations to enable the appropriate laboratory identification of VWD, in particular type 2B VWD.

  14. Poly (hydroxyethyl methacrylate-glycidyl methacrylate) films modified with different functional groups: In vitro interactions with platelets and rat stem cells.

    PubMed

    Bayramoglu, Gulay; Bitirim, Verda; Tunali, Yagmur; Arica, Mehmet Yakup; Akcali, Kamil Can

    2013-03-01

    Copolymerization of 2-hydroxyethylmethacrylate (HEMA) with glycidylmethacrylate (GMA) in the presence of α-α'-azoisobisbutyronitrile (AIBN) resulted in the formation of hydrogel films carrying reactive epoxy groups. Thirteen kinds of different molecules with pendant NH2 group were used for modifications of the p(HEMA-GMA) films. The NH2 group served as anchor binding site for immobilization of functional groups on the hydrogel film via direct epoxy ring opening reaction. The modified hydrogel films were characterized by FTIR, and contact angle studies. In addition, mechanical properties of the hydrogel films were studied, and modified hydrogel films showed improved mechanical properties compared with the non-modified film, but they are less elastic than the non-modified film. The biological activities of these films such as platelet adhesion, red blood cells hemolysis, and swelling behavior were studied. The effect of modified hydrogel films, including NH2, (using different aliphatic CH2 chain lengths) CH3, SO3H, aromatic groups with substituted OH and COOH groups, and amino acids were also investigated on the adhesion, morphology and survival of rat mesenchymal stem cells (MSCs). The MTT colorimetric assay reveals that the p(HEMA-GMA)-GA-AB, p(HEMA-GMA)-GA-Phe, p(HEMA-GMA)-GA-Trp, p(HEMA-GMA)-GA-Glu formulations have an excellent biocompatibility to promote the cell adhesion and growth. We anticipate that the fabricated p(HEMA-GMA) based hydrogel films with controllable surface chemistry and good stable swelling ratio may find extensive applications in future development of tissue engineering scaffold materials, and in various biotechnological areas.

  15. 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.

  16. 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.

  17. 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.

  18. Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

    PubMed

    Farquharson, Stuart; Shende, Chetan; Smith, Wayne; Huang, Hermes; Inscore, Frank; Sengupta, Atanu; Sperry, Jay; Sickler, Todd; Prugh, Amber; Guicheteau, Jason

    2014-12-21

    A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

  19. Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing.

    PubMed

    Eppley, Barry L; Woodell, Jennifer E; Higgins, Joel

    2004-11-01

    Growth factors released from activated platelets initiate and modulate wound healing in both soft and hard tissues. A recent strategy to promote the wound-healing cascade is to prepare an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma, that contains growth factors and administer it to wound sites. The purpose of this study was to quantitate platelet number and growth factors released from a prepared platelet concentrate. Whole blood was drawn from 10 healthy patients undergoing cosmetic surgery and concentrated into platelet-rich plasma. Platelet counts on whole blood and platelet-rich plasma were determined using a Cell-Dyn 3200. Platelet-derived growth factor-BB, transforming growth factor-beta1, vascular endothelial growth factor, endothelial growth factor, and insulin-like growth factor-1 were measured in the platelet-rich plasma using the enzyme-linked immunosorbent assay method. In addition, platelet activation during the concentration procedure was analyzed by measuring P selectin values in blood serum. An 8-fold increase in platelet concentration was found in the platelet-rich plasma compared with that of whole blood (baseline whole blood, 197 +/- 42 x 10 platelets/microl; platelet concentrate, 1600 +/- 330 x 10 platelets/microl). The concentration of growth factors also increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with platelet-rich plasma, the platelet-derived growth factor-BB concentration increased from 3.3 +/- 0.9 ng/ml to 17 +/- 8 ng/ml, transforming growth factor-beta1 concentration increased from 35 +/- 8 ng/ml to 120 +/- 42 ng/ml, vascular endothelial growth factor concentration increased from 155 +/- 110 pg/ml to 955 +/- 1030 pg/ml, and endothelial growth factor concentration increased from 129 +/- 61 pg/ml to 470 +/- 320 pg/ml. No increase was found for insulin-like growth factor-1. In addition, no

  20. A Randomized Phase II Trial Investigating the Effect of Platelet Function Inhibition on Circulating Tumor Cells in Patients With Metastatic Breast Cancer

    PubMed Central

    Roop, Ryan P.; Naughton, Michael J.; Van Poznak, Catherine; Schneider, Jochen G.; Lammers, Philip E.; Pluard, Timothy J.; Johnson, Farley; Eby, Charles S.; Weilbaecher, Katherine N.

    2014-01-01

    Background Blockade of platelet activation and aggregation can inhibit metastasis in preclinical models and is associated with cancer prevention. To test whether disruption of platelet function with clopidogrel and aspirin would decrease the number of circulating tumor cells (CTCs) in patients with metastatic breast cancer, a randomized phase II study was performed. Methods Patients with metastatic breast cancer who were not currently receiving cytotoxic chemotherapy were eligible. Patients were randomized to receive either clopidogrel and aspirin or to a control group receiving no treatment. Phlebotomy was performed at baseline, at 2 and 4 weeks, and monthly thereafter to obtain specimens to assess CTC, platelet aggregation, and thrombin activity. The primary end point was the proportion of patients with detectable CTCs at 1 month. Results Forty-eight patients were enrolled and 42 were evaluable at 1 month. Baseline CTC numbers were ≥ 5 in 13% and ≥ 1 in 65% of patients. Despite adequate platelet function inhibition in the treatment group, the proportion of patients with detectable CTCs was similar between the clopidogrel/aspirin and control groups at baseline (P = .21) and 4 weeks (P = .75), showing no treatment effect. Measured endogenous thrombin potential did not correlate with CTC number. No bleeding-related serious adverse events (SAEs) occurred. Conclusion The baseline CTC numbers were lower than expected, decreasing the ability to detect an impact of platelet inhibition on CTCs. Clopidogrel and aspirin were well tolerated. Future studies evaluating the potential therapeutic role of antiplatelet therapy in breast cancer remain of interest, and they may be informed by these results. PMID:24267729

  1. Platelet-Rich Plasma Increases Growth and Motility of Adipose Tissue-Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function.

    PubMed

    D'Esposito, Vittoria; Passaretti, Federica; Perruolo, Giuseppe; Ambrosio, Maria Rosaria; Valentino, Rossella; Oriente, Francesco; Raciti, Gregory A; Nigro, Cecilia; Miele, Claudia; Sammartino, Gilberto; Beguinot, Francesco; Formisano, Pietro

    2015-10-01

    Adipose tissue-derived mesenchymal stem cells (Ad-MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet-rich plasma (PRP) on Ad-MSC and adipocyte function. PRP increased Ad-MSC viability, proliferation rate and G1-S cell cycle progression, by at least 7-, 2-, and 2.2-fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad-MSC growth. PRP also accelerated cell migration by at least 1.5-fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR-γ and AP-2 mRNAs, while it increased leptin production by 3.5-fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)-6, IL-8, IL-10, Interferon-γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad-MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro-angiogenic factors, which may facilitate tissue regeneration processes.

  2. Effects of tirofiban on platelet activation and endothelial function in patients with ST-elevation myocardial infarction undergoing primary percutaneous coronary intervention.

    PubMed

    Wang, Kuan; Zuo, Guoxing; Zheng, Liuying; Zhang, Cheng; Wang, Dong; Cao, Zhongnan; Hu, Sheng; Du, Xinping

    2015-01-01

    This pilot study examined, for the first time, the effect of intracoronary administration of tirofiban, an inhibitor of platelet aggregation, on platelet activation and endothelial dysfunction in patients with ST-segment-elevated myocardial infarction (STEMI) undergoing percutaneous coronary intervention (PCI). A total of 119 STEMI patients were randomized into either tirofiban group (n = 72, intracoronary injection of 10 μg/kg tirofiban prior to PCI, followed by intravenous infusion at 0.15 μg/kg min) or a control group (n = 47), which did not receive tirofiban. Periprocedural administration of tirofiban was associated with significantly reduced levels of platelet activation (lower levels of CD62P and PAC-1) and endothelial dysfunction (reduced levels of endothelial microparticles, VCAM-1, and ICAM-1) 48 h after PCI. At 10 days after PCI, patients in the tirofiban group had a higher incidence of complete STR (78.7 vs. 65.0%) and higher left ventricular ejection fractions (47.8 vs. 44.2) compared to those in the control group. The clinical outcomes between two groups did not differ significantly two weeks after treatment. The results demonstrated that periprocedural administration of tirofiban is associated with significantly attenuated platelet activation and endothelial dysfunction in STEMI patients undergoing PCI. This may have contributed to the improved myocardial reperfusion and preservation of left ventricular systolic function in these patients.

  3. Platelets: production, morphology and ultrastructure.

    PubMed

    Thon, Jonathan N; Italiano, Joseph E

    2012-01-01

    Platelets are anucleate, discoid cells, roughly 2-3 μm in diameter that function primarily as regulators of hemostasis, but also play secondary roles in angiogensis and innate immunity. Although human adults contain nearly one trillion platelets in circulation that are turned over every 8-10 days, our understanding of the mechanisms involved in platelet production is still incomplete. Platelets stem from large (30-100 μm) nucleated cells called megakaryocytes that reside primarily in the bone marrow. During maturation megakaryocytes extend long proplatelet elongations into sinusoidal blood vessels from which platelets ultimately release. During this process, platelets develop a number of distinguishable structural elements including: a delimited plasma membrane; invaginations of the surface membrane that form the open canalicular system (OCS); a closed-channel network of residual endoplasmic reticulum that form the dense tubular system (DTS); a spectrin-based membrane skeleton; an actin-based cytoskeletal network; a peripheral band of microtubules; and numerous organelles including α-granules, dense-granules, peroxisomes, lysosomes, and mitochondria. Proplatelet elongation and platelet production is an elaborate and complex process that defines the morphology and ultrastructure of circulating platelets, and is critical in understanding their increasingly numerous and varied biological functions.

  4. Deciphering the human platelet sheddome

    PubMed Central

    Fong, Karen P.; Barry, Colin; Tran, Anh N.; Traxler, Elizabeth A.; Wannemacher, Kenneth M.; Tang, Hsin-Yao; Speicher, Kaye D.; Blair, Ian A.; Speicher, David W.; Grosser, Tilo

    2011-01-01

    Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function. PMID:20962327

  5. 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.

  6. 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

  7. Development And Application of Functional Assays For Freshwater Dissolved Organic Matter

    NASA Astrophysics Data System (ADS)

    Thacker, S.; Tipping, E.; Gondar, D.; Baker, A.

    2006-12-01

    Dissolved organic matter (DOM) in natural waters participates in many important ecological and geochemical reactions, including acid-base buffering, light absorption, proton binding, binding of heavy metals, organic contaminants, aluminium and radionuclides, adsorption at surfaces, aggregation and photochemical reactivity. We are studying DOM in order to understand and quantify these functional properties, so we can use the knowledge to predict the influence of DOM on the natural freshwater environment. As DOM has no readily identifiable structure, our approach is to measure what it does, rather than what it is. Thus, we have developed a series of 12 standardised, reproducible assays of physico-chemical functions of dissolved organic matter (DOM) in freshwaters. The assays provide quantitative information on light absorption, fluorescence, photochemical fading, pH buffering, copper binding, benzo(a)pyrene binding, hydrophilicity and adsorption to alumina. We have collected twenty DOM samples in total, ten samples from a eutrophic lake (Esthwaite Water) and ten samples from three stream waters. A mild isolation method was then used to concentrate the DOM samples for the assay work. When assaying the concentrates, parallel assays were also preformed with Suwannee River Fulvic Acid (SRFA), as a quality control standard. Our results showed that; (i) for eleven of the assays, the variability among the twenty DOM samples was significantly (p<0.001) greater than can be explained by analytical error, i.e. by comparison with results from the SRFA quality control; (ii) the functional properties of the DOM from Esthwaite Water are strongly influenced by the seasonally-dependent input of autochthonous DOM, derived from phytoplankton. The autochthonous DOM is less fluorescent, light absorbing, hydrophobic and has a lower acid group content and capacity to be adsorbed onto alumina than terrestrially derived allochthonous DOM; (iii) significant correlations were found between

  8. Activated platelets inhibit hepatocellular carcinoma cell differentiation and promote tumor progression via platelet-tumor cell binding

    PubMed Central

    Xu, Jingchao; Li, Bing; Liu, Yue-Jian; Cheng, Cheng; Zhou, Chunyan; Zhao, Yongfu; Liu, Yang

    2016-01-01

    Lack of differentiation in hepatocellular carcinoma (HCC) is associated with increased circulating platelet size. We measured platelet activation and plasma adenosine diphosphate (ADP) levels in HCC patients based on differentiation status. Local platelet accumulation and platelet-hepatoma cell binding were measured using immunohistochemistry (IHC) or flow cytometry. Using a xenograft assay in NON/SCID mice, we tested the effects of the anti-platelet drug clopidogrel on platelet activation, platelet infiltration, platelet-tumor cell binding and tumor cell differentiation. HCC patients with poor differentiation status displayed elevated platelet activation and higher ADP levels. Platelets accumulated within poorly differentiated tissues and localized at hepatoma cell membranes. Platelet-tumor cell binding was existed in carcinoma tissues, largely mediated by P-selectin on platelets. NOD/SCID mice with xenograft tumors also exhibited increased platelet activation and platelet-tumor cell binding. Clopidogrel therapy triggered hepatoma cell differentiation by attenuating platelet activation and platelet-tumor cell binding. TCF4 knockdown promoted HepG-2 cell differentiation and inhibited tumor formation, and TCF4 could be the potential downstream target for clopidogrel therapy. PMID:27542264

  9. Optimal Same-Day Platelet Inhibition in Patients Receiving Drug-Eluting Stents With or Without Previous Maintenance Thienopyridine Therapy: from the Evaluation of Platelet Inhibition in Patients Having A VerifyNow Assay (EPIPHANY) Trial.

    PubMed

    Lee, Michael S; Shlofmitz, Evan; Haag, Elizabeth; Santiago, Lyn; Pollack, Simcha; Reichek, Nathaniel; Shlofmitz, Richard A

    2017-04-01

    We determined if high on-treatment platelet reactivity (HTPR) can be overcome on the day of percutaneous coronary intervention (PCI) in patients with or without previous maintenance thienopyridine therapy. Patients with HTPR, as defined as P2Y12 reaction units (PRU) >230, were switched to an alternate thienopyridine. Patients with HTPR undergoing PCI are at increased risk for ischemic complications. A total of 429 patients undergoing PCI with drug-eluting stents were enrolled. Patients on maintenance thienopyridine (n = 249) with PRU >230 were loaded with the alternative thienopyridine. Patients who were thienopyridine naïve (n = 180) were randomized to clopidogrel 600 (n = 90) or prasugrel 60 mg (n = 90). Patients with HTPR were loaded with the alternative agent. Patients on maintenance clopidogrel (n = 192) had a higher prevalence of HTPR compared with prasugrel (n = 57; 51% vs 4%, p <0.001). Patients on maintenance clopidogrel with HTPR (n = 98) who were loaded with prasugrel achieved PRU ≤230 in 97%. Thienopyridine-naïve patients loaded with clopidogrel had a higher prevalence of HTPR compared with prasugrel (37% vs 3%, p <0.001). Clopidogrel-loaded patients with HTPR (n = 33) who were reloaded with prasugrel achieved PRU ≤230 in 94%. All 3 prasugrel-loaded patients with HTPR treated with clopidogrel achieved PRU ≤230. Two patients experienced 30-day major adverse clinical events. One patient experienced Thrombolysis In Myocardial Infarction major bleeding. In conclusion, HTPR can be overcome in patients with and without previous maintenance thienopyridine therapy by identifying patients with HTPR and switching to an alternate thienopyridine.

  10. 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

  11. [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.

  12. Platelet dysfunction associated with Wilms tumor and hyaluronic acid.

    PubMed

    Bracey, A W; Wu, A H; Aceves, J; Chow, T; Carlile, S; Hoots, W K

    1987-03-01

    Acquired von Willebrand disease (AVWD) has been described in two cases of nephroblastoma. We studied a patient with nephroblastoma who presented with a coagulopathy suggestive of AVWD. The subject had undetectable levels of F.VIIIR:Ag, diminished F.VIIIR:WF (16.3%), F.VIII:C activity (37%), and lack of platelet aggregation to ADP, epinephrine, collagen, and arachidonic acid. These results were associated with abnormally high serum levels (850 mg/dl) of hyaluronic acid (HA), which made the patient's serum hyperviscous. Examination of the neoplasm revealed HA in the tumor matrix. All abnormalities of coagulation resolved after chemotherapy and extirpation of the neoplasm, which produced normal serum HA levels. To study the effects of HA on platelet function, we added HA to normal platelet-poor plasma (NPP), which rendered F.VIIIR:Ag undetectable; treatment of HA with hyaluronidase eliminated F.VIIIR:Ag assay interference caused by HA. F.VIII:C activity decreased in vitro when HA was mixed with normal platelet-poor plasma (NPP). HA reduced the initial slope of normal platelet aggregation. Partial correction of platelet aggregation occurred after hyaluronidase treatment of HA-spiked PRP. Experiments in rabbits exposed to HA (serum level 400 mg/dl) demonstrated abnormalities similar to those noted in the patient. Shear rate studies of whole blood containing HA (500 mg/dl) yielded high shear stress, 27-136 dynes/cm2 over shear rates of 10-216 sec-1. We conclude that the coagulopathy demonstrated in this case is secondary to hyperviscosity produced by elevated levels of HA, which interferes with the assay for F.VIIIR:Ag. Thus the acquired coagulopathy associated with other cases of nephroblastoma may present as spurious von Willebrand disease.

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

    SciTech Connect

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

    1990-07-15

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

  14. Electrochemical and anticorrosion behaviors of hybrid functionalized graphite nano-platelets/tripolyphosphate in epoxy-coated carbon steel

    SciTech Connect

    Mohammadi, Somayeh; Shariatpanahi, Homeira; Taromi, Faramarz Afshar; Neshati, Jaber

    2016-08-15

    Highlights: • FGNP was combined with TPP to obtain a hybrid nano-particle. • TEM image showed uniform distribution of the hybrid nanoparticles in epoxy coating. • FGNP is a substrate for linking of TPP anions by hydrogen bonding. • FGNP as an accelerator, provides rapid iron phosphate passive film formation. • The hybrid nano-particle can provide long-term corrosion protection. - Abstract: Functionalized graphite nano-platelets (FGNP) were combined with tripolyphosphate (TPP) to gain a hybrid nano-particle (FGNP-TPP) with homogenous dispersion in epoxy, resulting in an excellent anti-corrosion coating for carbon steel substrate. Characterization analyses of the hybrid nano-particle were performed by FT-IR, SEM, XRD and TEM. TPP was linked to FGNP nano-particles by hydrogen bondings. Different epoxy coatings formulated with 1 wt.% of FGNP, FGNP-TPP and TPP were evaluated. Electrochemical investigations, salt spray and pull-off tests showed that the hybrid nano-particle can provide long-term corrosion protection compared to FGNP and TPP due to synergistic effect between FGNP as an accelerator and TPP as a corrosion inhibitor to produce a uniform and stable iron-phosphate passive film with high surface coverage.

  15. Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma.

    PubMed

    Muqaku, Besnik; Eisinger, Martin; Meier, Samuel M; Tahir, Ammar; Pukrop, Tobias; Haferkamp, Sebastian; Slany, Astrid; Reichle, Albrecht; Gerner, Christopher

    2017-01-01

    Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

  16. Functional study of a monoclonal antibody to IgE Fc receptor (Fc epsilon R2) of eosinophils, platelets, and macrophages

    PubMed Central

    1986-01-01

    An IgM mAb (BB10) was produced by immunization of mice with human eosinophils purified according to their abnormal low density ("hypodense" cells), and previously shown to exhibit increased IgE- dependent antiparasite cytotoxicity. This BB10 antibody, selected for positive fluorescence staining of hypodense blood or lung eosinophils and low or negative staining of normodense eosinophils or neutrophils, could strongly inhibit IgE-dependent cytotoxicity of human eosinophils and platelets. The specificity for the IgE Fc receptor was suggested by the high levels of inhibition of IgE rosettes formed by eosinophils after incubation with the purified IgM fraction of BB10, whereas other receptors (Fc gamma R, CR1) were not affected. On the other hand, BB10, able to inhibit rat eosinophil Fc epsilon R, did not react with the IgE Fc receptor on mast cells or basophils. A technique using radioiodinated BB10 allowed us to quantify the specific binding of BB10 to human eosinophils and platelets. Competition experiments revealed a crossinhibition between the binding of BB10 and IgE, suggesting the specificity of BB10 for the IgE binding site of eosinophil, platelet, and monocyte Fc epsilon R. Three proteins having extrapolated Mr of 32,000, 43,000-45,000, and 97,000 were found in the platelet extract eluted from a BB10 or from an IgE immunosorbent column. These findings confirm the similarities between IgE Fc receptors on human eosinophils, platelets, and macrophages, already observed with polyclonal antibodies directed against the B lymphocyte Fc epsilon receptor. They suggest, moreover, that the mAb BB10 can represent a good reagent for further investigations on the structure and the functions of this IgE Fc receptor (Fc epsilon R2). PMID:2425032

  17. Functional analysis of human BRCA2 variants using a mouse embryonic stem cell-based assay.

    PubMed

    Kuznetsov, Sergey G; Chang, Suhwan; Sharan, Shyam K

    2010-01-01

    We describe here a comprehensive and reliable assay to test the functional significance of variants of unknown clinical significance (VUS) identified in the human breast cancer susceptibility gene, BRCA2. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse embryonic stem cells. The procedure involves generation of a desired mutation in BRCA2 present in a bacterial artificial chromosome (BAC) and the introduction of the BAC into ES cells engineered for the assay. These ES cells have one null and one conditional allele of Brca2. First, the effect of the BRCA2 variants on the viability of ES cells is tested by Cre-mediated deletion of the conditional allele. Subsequently, variants that result in viable ES cells are examined for their effect on known functions of BRCA2 using a variety of functional assays such as sensitivity to genotoxic agents, in vivo and in vitro proliferation, effect on homologous recombination and genomic stability. The method described herein allows for the analysis of three to five sequence variants within 2-3 months. This approach can also be used for functional analysis of variants identified in other human disease genes that result in a phenotype detectable in ES cells.

  18. 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

  19. The Hermansky-Pudlak syndrome 1 (HPS1) and HPS2 genes independently contribute to the production and function of platelet dense granules, melanosomes, and lysosomes.

    PubMed

    Feng, Lijun; Novak, Edward K; Hartnell, Lisa M; Bonifacino, Juan S; Collinson, Lucy M; Swank, Richard T

    2002-03-01

    Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease affecting the related subcellular organelles platelet dense granules, lysosomes, and melanosomes. The mouse genes for HPS, pale ear and pearl, orthologous to the human HPS1 and HPS2 (ADTB3A) genes, encode a novel protein of unknown function and the beta(3)A subunit of the AP-3 adaptor complex, respectively. To test for in vivo interactions between these genes in the production and function of intracellular organelles, mice doubly homozygous for the 2 mutant genes were produced by appropriate breeding. Cooperation between the 2 genes in melanosome production was evident in increased hypopigmentation of the coat together with dramatic quantitative and qualitative alterations of melanosomes of the retinal pigment epithelium and choroid of double mutant mice. Lysosomal and platelet dense granule abnormalities, including hyposecretion of lysosomal enzymes from kidneys and depression of serotonin concentrations of platelet dense granules were likewise more severe in double than single mutants. Also, lysosomal enzyme concentrations were significantly increased in lungs of double mutant mice. Interaction between the 2 genes was specific in that effects on organelles were confined to melanosomes, lysosomes, and platelet dense granules. Together, the evidence indicates these 2 HPS genes function largely independently at the whole organism level to affect the production and function of all 3 organelles. Further, the increased lysosomal enzyme levels in lung of double mutant mice suggest a cause of a major clinical problem of HPS, lung fibrosis. Finally, doubly mutant HPS mice are a useful laboratory model for analysis of severe HPS phenotypes.

  20. Biologic nanoparticles and platelet reactivity

    PubMed Central

    Miller, Virginia M; Hunter, Larry W; Chu, Kevin; Kaul, Vivasvat; Squillace, Phillip D; Lieske, John C; Jayachandran, Muthuvel

    2009-01-01

    Aim Nanosized particles (NPs) enriched in hydroxyapatite and protein isolated from calcified human tissue accelerate occlusion of endothelium-denuded arteries when injected intravenously into rabbits. Since platelet aggregation and secretory processes participate in normal hemostasis, thrombosis and vascular remodeling, experiments were designed to determine if these biologic NPs alter specific platelet functions in vitro. Methods Platelet-rich plasma was prepared from citrate anticoagulated human blood. Platelet aggregation and ATP secretion were monitored in response to thrombin receptor agonists peptide (10 μM) or convulxin (50 μg/ml) prior to and following 15 min incubation with either control solution, human-derived NPs, bovine-derived NPs or crystals of hydroxyapatite at concentrations of 50 and 150 nephelometric turbidity units. Results Incubation of platelets for 15 min with either human- or bovine-derived NPs reduced aggregation induced by thrombin receptor activator peptide and convulxin in a concentration-dependent manner. Hydroxyapatite caused a greater inhibition than either of the biologically derived NPs. Human-derived NPs increased ATP secretion by unstimulated platelets during the 15 min incubation period. Conclusion Effects of bovine-derived and hydroxyapatite NPs on basal release of ATP were both time and concentration dependent. These results suggest that biologic NPs modulate both platelet aggregation and secretion. Biologically derived NPs could modify platelet responses within the vasculature, thereby reducing blood coagulability and the vascular response to injury. PMID:19839809

  1. 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

  2. Contact- and agonist-regulated microvesiculation of human platelets.

    PubMed

    Zhang, Yanjun; Liu, Xiao; Liu, Li; Zaske, Ana-Maria; Zhou, Zhou; Fu, Yuanyuan; Yang, Xi; Conyers, Jodie L; Li, Min; Dong, Jing-fei; Zhang, Jianning

    2013-08-01

    After exposure to an agonist, platelets are activated and become aggregated. They also shed membrane microparticles that participate in the pathogenesis of thrombosis, hyper-coagulation and inflammation. However, microvesiculation can potentially disrupt the integrity of platelet aggregation by shedding the membrane receptors and phosphatidylserine critical for forming and stabilising a platelet clot. We tested the hypothesis that adhesion and microvesiculation are functions of different subsets of platelets at the time of haemostasis by real-time monitoring of agonist-induced morphological changes and microvesiculation of human platelets.We identified two types of platelets that are adherent to fibrinogen: a high density bubble shape (HDBS) and low-density spread shape (LDSS). Adenosine diphosphate (ADP) predominantly induced HDBS platelets to vesiculate, whereas LDSS platelets were highly resistant to such vesiculation. Thrombin-receptor activating peptide (TRAP) stabilised platelets against microvesiculation by promoting a rapid HDBS-to-LDSS morphological transition. These activities of ADP and TRAP were reversed for platelets in suspension, independent of an engagement integrin αIIbβ3. As the result of membrane contact, LDSS platelets inhibited the microvesiculation of HDBS platelets in response to ADP. Aspirin and clopidogrel inhibited ADP-induced microvesiculation through different mechanisms. These results suggest that platelet aggregation and microvesiculation occur in different subsets of platelets and are differently regulated by agonists, platelet-platelets and platelet-fibrinogen interactions.

  3. Identification and Functional Characterization of Glycosylation of Recombinant Human Platelet-Derived Growth Factor-BB in Pichia pastoris.

    PubMed

    Dai, Mengmeng; Yu, Changming; Fang, Ting; Fu, Ling; Wang, Jing; Zhang, Jun; Ren, Jun; Xu, Junjie; Zhang, Xiaopeng; Chen, Wei

    2015-01-01

    Yeast Pichia pastoris is a widely used system for heterologous protein expression. However, post-translational modifications, especially glycosylation, usually impede pharmaceutical application of recombinant proteins because of unexpected alterations in protein structure and function. The aim of this study was to identify glycosylation sites on recombinant human platelet-derived growth factor-BB (rhPDGF-BB) secreted by P. pastoris, and investigate possible effects of O-linked glycans on PDGF-BB functional activity. PDGF-BB secreted by P. pastoris is very heterogeneous and contains multiple isoforms. We demonstrated that PDGF-BB was O-glycosylated during the secretion process and detected putative O-glycosylation sites using glycosylation staining and immunoblotting. By site-directed mutagenesis and high-resolution LC/MS analysis, we, for the first time, identified two threonine residues at the C-terminus as the major O-glycosylation sites on rhPDGF-BB produced in P. pastoris. Although O-glycosylation resulted in heterogeneous protein expression, the removal of glycosylation sites did not affect rhPDGF-BB mitogenic activity. In addition, the unglycosylated PDGF-BBΔGly mutant exhibited the immunogenicity comparable to that of the wild-type form. Furthermore, antiserum against PDGF-BBΔGly also recognized glycosylated PDGF-BB, indicating that protein immunogenicity was unaltered by glycosylation. These findings elucidate the effect of glycosylation on PDGF-BB structure and biological activity, and can potentially contribute to the design and production of homogeneously expressed unglycosylated or human-type glycosylated PDGF-BB in P. pastoris for pharmaceutical applications.

  4. The PFA-100R cannot detect blood group-dependent inhibition of platelet function by eptifibatide or abciximab at therapeutic plasma concentrations.

    PubMed

    Feuring, M; Ruf, A; Schultz, A; Wehling, M

    2010-01-01

    Previous investigations revealed that AB0 blood groups are associated with divergent concentrations of several coagulation factors. Concentrations of von Willebrand factor (vWF) and factor VIII are lower in individuals with blood group 0 compared to subjects with blood group A, B or AB, which might in turn result in a reduced inhibition of platelet aggregation in individuals with blood group 0. The aim of the present in vitro investigation was to elucidate the impact of AB0 blood group-dependent vWF concentrations on eptifibatide and abciximab mediated inhibition of GPIIb/IIIa function. Platelet function was measured with the platelet function analyzer PFA-100(R) at baseline and at increasing concentrations of eptifibatide and abciximab. It was stratified for blood group 0 vs A. If measured with the collagen/ADP cartridge, blood group 0 was associated with a prolonged mean baseline closure time in comparison with blood group A (94.3 +/- 14.6 s vs. 74.6 +/- 9.9 s, p = 0.007) which was paralleled by reduced concentrations of vWF and factor VIII. In contrast, no statistically significant differences in closure times (167.4 +/- 83.9 s vs. 140.1 +/- 99.0 s, p = 0.562) could be found in the presence of eptifibatide (0.1 microg/ml). Higher concentrations of abciximab (1 microg/ml) than those of eptifibatide were needed to increase the closure times in both cartridges of the PFA-100, but at this concentration of abciximab differences in closure times could not be detected most probably due to higher variability at these drug concentrations. The PFA-100(R) is not suitable for monitoring abciximab or eptifibatide within the therapeutic concentration range because the highest concentrations where the PFA-100(R) had measurable closure times of below 300 s is much too low to lead to the necessary platelet inhibition and, consequently, does not resemble the in vivo situation.

  5. Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

    PubMed Central

    D’Souza, Ashwin; Sanghavi, Paulomi; Rai, Ashim; Pathak, Divya; Mallik, Roop

    2017-01-01

    We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance. PMID:28239623

  6. A simple test for lacrimal gland function: a tear lactoferrin assay by radial immunodiffusion.

    PubMed

    Janssen, P T; van Bijsterveld, O P

    1983-01-01

    A radial immunodiffusion assay for tear lactoferrin is described. Tear samples, collected on filter paper discs, could be applied directly to immunodiffusion plates after blotting. In 58 tear samples from healthy subjects an average lactoferrin concentration of 1.42 g/l was found. A high degree of correlation was found between the lactoferrin radial immunodiffusion and the lysozyme agar diffusion assay in a heterogeneous group, ranging from healthy subjects to patients with severe keratoconjunctivitis sicca. A lower limit of normal (0.78 g/l) could be determined for the tear lactoferrin concentration. The assay does not require laboratory facilities and therefore is an easily accessible alternative for or an addition to lacrimal gland function tests currently used.

  7. 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

  8. 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.

  9. 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

  10. Genotyping for human platelet alloantigen polymorphisms: applications in the diagnosis of alloimmune platelet disorders.

    PubMed

    Curtis, Brian R

    2008-09-01

    Molecular typing for platelet allelic polymorphisms was first made possible by discovery of the HPA-1a/1b single nucleotide polymorphism in 1989. Since then, six other biallelic human platelet antigen (HPA) systems have been determined and can be typed using genomic DNA. The introduction of polymerase chain reaction enabled development of several different assays including polymerase chain reaction-sequence-specific primer, melting curve analysis by LightCycler, and 5'-nuclease assays. More recently, multiplex polymerase chain reaction has allowed for the development of high-throughput assays for genotyping large numbers of patients and blood donors for not only platelet gene polymorphisms but also for those of other blood cell genes. Platelet genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera to complement the clinical history in the diagnosis of alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura, and multiplatelet transfusion refractoriness. In addition, it has made possible prenatal platelet typing of the fetus in suspected cases of FNAIT and large-scale blood donor typing for provision of antigen-negative platelets to transfuse highly alloimmunized patients. Platelet genotyping may also someday prove important as an aid in determining the relative risk of patients for various thrombotic disorders.

  11. 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.

  12. Evidence of platelet activation in multiple sclerosis

    PubMed Central

    Sheremata, William A; Jy, Wenche; Horstman, Lawrence L; Ahn, Yeon S; Alexander, J Steven; Minagar, Alireza

    2008-01-01

    Objective A fatality in one multiple sclerosis (MS) patient due to acute idiopathic thrombocytopenic purpura (ITP) and a near fatality in another stimulated our interest in platelet function abnormalities in MS. Previously, we presented evidence of platelet activation in a small cohort of treatment-naive MS patients. Methods In this report, 92 normal controls and 33 stable, untreated MS patients were studied. Platelet counts, measures of platelet activation [plasma platelet microparticles (PMP), P-selectin expression (CD62p), circulating platelet microaggragtes (PAg)], as well as platelet-associated IgG/IgM, were carried out. In addition, plasma protein S activity was measured. Results Compared to controls, PMP were significantly elevated in MS (p < 0.001) and CD62p expression was also markedly elevated (p < 0.001). Both are markers of platelet activation. Platelet-associated IgM, but not IgG, was marginally elevated in MS (p = 0.01). Protein S in MS patients did not differ significantly from normal values. Conclusion Platelets are significantly activated in MS patients. The mechanisms underlying this activation and its significance to MS are unknown. Additional study of platelet activation and function in MS patients is warranted. PMID:18588683

  13. Functional assays for analysis of variants of uncertain significance in BRCA2

    PubMed Central

    Guidugli, Lucia; Carreira, Aura; Caputo, Sandrine M.; Ehlen, Asa; Galli, Alvaro; Monteiro, Alvaro N.A.; Neuhausen, Susan L.; Hansen, Thomas V.O.; Couch, Fergus J.; Vreeswijk, Maaike P.G.

    2014-01-01

    Missense variants in the BRCA2 gene are routinely detected during clinical screening for pathogenic mutations in patients with a family history of breast and ovarian cancer. These subtle changes frequently remain of unknown clinical significance because of the lack of genetic information that may help establish a direct correlation with cancer predisposition. Therefore alternative ways of predicting the pathogenicity of these variants are urgently needed. Since BRCA2 is a protein involved in important cellular mechanisms such as DNA repair, replication and cell cycle control, functional assays have been developed that exploit these cellular activities to explore the impact of the variants on protein function. In this review we summarize assays developed and currently utilized for studying missense variants in BRCA2. We specifically depict details of each assay, including VUS analyzed, and describe a validation set of (genetically) proven pathogenic and neutral missense variants to serve as a golden standard for the validation of each assay. Guidelines are proposed to enable implementation of laboratory-based methods to assess the impact of the variant on cancer risk. PMID:24323938

  14. 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.

  15. Real-time monitoring of the functional status of platelets treated by Infukoll using a computer-aided laser phase microscope

    NASA Astrophysics Data System (ADS)

    Vasilenko, Irina A.; Babakova, Svetlana; Kiseleva, Elena; Dyugeev, Adyan; Konradov, Alexander A.; Shabalin, Vladimir

    1999-02-01

    Combined analysis of optic-geometrical characteristics makes it possible to comprehensively evaluate the morphological and functional state of the cytological object, which can not be done during visual observation. The technique is discussed for real-time monitoring of the functional status of platelets using computer-aided phase microscope (CPM) 'Cytoscan'. High accuracy and sensitivity of CPM with respect to determination of local temporal phase make it possible to register the dynamic processes in the voluntarily chosen points and sections of micro-objects, to obtain the Fourier's spectra and other characteristics suitable for statistical analysis. Human platelets were prepared from venous blood of healthy donors and pregnant women by standard methods, suspended in culture medium 199 and treated by different doses of 6% Infukoll HES. Nonfixating and nonstaining cells were studied with CPM: height accuracy 0.5 nm, magnification 1000, acquisition time 4 - 30 s. In our experiments we used time resolution about 0.03 s and 30 x lens with numerical aperture 0.65. During investigations of temporal processes a certain section was chosen in the topogram of cell image and local values for the phase of scattered wave in each of the points of the chosen cell's profile were measured. On the basis of the results of automated phase image analysis of optic-geometrical characteristics of living cells, the new quantitative express-method for evaluating of the functional status of human platelets was developed and tested. The structural changes of cells were visualized in alteration of 3-D images, phase profiles, in the decrease of mean cell phase diameters, heights, volumes, in disturbance of histograms of phase heights distribution by cell image points. New data on the behavior of platelets treated by Infukoll in vitro and in vivo were obtained. Analysis of intracellular dynamics was allowed to characterize the cell's regions of maximal activity, but the intensity of processes

  16. [Platelet allo-antibodies identification strategies for preventing and managing platelet refractoriness].

    PubMed

    Basire, A; Picard, C

    2014-11-01

    Platelet refractoriness is a serious complication for patients receiving recurrent platelet transfusions, which can be explained by non-immune and immune causes. Human Leukocyte Antigens (HLA) allo-immunization, especially against HLA class I, is the major cause for immune platelet refractoriness. To a lesser extent, allo-antibodies against specific Human Platelet Antigen (HPA) are also involved. Pregnancy, transplantation and previous transfusions can lead to allo-immune reaction against platelet antigens. After transfusion, platelet count is decreased by accelerated platelet destruction related to antibodies fixation on incompatible platelet antigens. New laboratory tests for allo-antibodies identification were developed to improve sensibility and specificity, especially with the LUMINEX(®) technology. The good use and interpretation of these antibodies assays can improve strategies for platelet refractoriness prevention and management with a patient adapted response. Compatible platelets units can be selected according to their identity with recipient typing or immune compatibility regarding HLA or HPA antibodies or HLA epitope compatibility. Prospective studies are needed to further confirm the clinical benefit of new allo-antibodies identification methods and consensus strategies for immune platelet refractoriness management.

  17. Pulmonary function in patients with acute coronary syndrome treated with ticagrelor or clopidogrel (from the Platelet Inhibition and Patient Outcomes [PLATO] pulmonary function substudy).

    PubMed

    Storey, Robert F; Becker, Richard C; Harrington, Robert A; Husted, Steen; James, Stefan K; Cools, Frank; Steg, Philippe Gabriel; Khurmi, Nardev S; Emanuelsson, Hakan; Lim, Soo Teik; Cannon, Christopher P; Katus, Hugo A; Wallentin, Lars

    2011-12-01

    The Platelet Inhibition and Patient Outcomes (PLATO) trial showed that ticagrelor reduced the risk for cardiovascular events in patients with acute coronary syndromes compared to clopidogrel but was associated with increased incidence of dyspnea. This substudy assessed whether ticagrelor affects pulmonary function in patients with acute coronary syndromes: 199 patients enrolled in the PLATO trial and receiving randomized treatment with ticagrelor 90 mg twice daily (n = 101) or clopidogrel 75 mg/day (n = 98) took part in the pulmonary function substudy. Patients with advanced lung disease, congestive heart failure, or coronary artery bypass graft surgery after the index event were excluded. Pulse oximetry (blood oxygen saturation), spirometry (forced expiratory volume in 1 second, forced vital capacity, and forced expiratory flow between 25% and 75% of forced vital capacity before and 20 minutes after inhalation of a β(2) agonist), lung volumes (total lung capacity, functional residual capacity, residual volume), and diffusion capacity were performed after patients received study medication for 30 to 40 days. Tests were then repeated <10 days before and approximately 30 days after the discontinuation of study medication. After a mean treatment duration of 31 days, there were no differences between the groups for any of the pulmonary function parameters. At the end of treatment (mean 211 days) and after the discontinuation of study medication (mean 32 days after the last dose), there was also no evidence of a change in pulmonary function in either group. For example, forced expiratory volume in 1 second values before β(2) agonist inhalation in the ticagrelor and clopidogrel groups were 2.81 ± 0.73 and 2.70 ± 0.84 L, respectively, at the first visit and did not change significantly at subsequent visits. In conclusion, no effect of ticagrelor on pulmonary function was seen in this cohort of patients with acute coronary syndromes compared to clopidogrel.

  18. A radial basis function (RBF) finite difference method for the simulation of reaction-diffusion equations on stationary platelets within the augmented forcing method

    NASA Astrophysics Data System (ADS)

    Shankar, Varun; Wright, Grady B.; Fogelson, Aaron L.; Kirby, Robert M.

    2014-05-01

    We present a computational method for solving the coupled problem of chemical transport in a fluid (blood) with binding/unbinding of the chemical to/from cellular (platelet) surfaces in contact with the fluid, and with transport of the chemical on the cellular surfaces. The overall framework is the Augmented Forcing Point Method (AFM) (\\emph{L. Yao and A.L. Fogelson, Simulations of chemical transport and reaction in a suspension of cells I: An augmented forcing point method for the stationary case, IJNMF (2012) 69, 1736-52.}) for solving fluid-phase transport in a region outside of a collection of cells suspended in the fluid. We introduce a novel Radial Basis Function-Finite Difference (RBF-FD) method to solve reaction-diffusion equations on the surface of each of a collection of 2D stationary platelets suspended in blood. Parametric RBFs are used to represent the geometry of the platelets and give accurate geometric information needed for the RBF-FD method. Symmetric Hermite-RBF interpolants are used for enforcing the boundary conditions on the fluid-phase chemical concentration, and their use removes a significant limitation of the original AFM. The efficacy of the new methods are shown through a series of numerical experiments; in particular, second order convergence for the coupled problem is demonstrated.

  19. Signaling during platelet adhesion and activation

    PubMed Central

    Li, Zhenyu; Delaney, M. Keegan; O’Brien, Kelly A.; Du, Xiaoping

    2011-01-01

    Upon vascular injury, platelets are activated by adhesion to adhesive proteins like von Willebrand factor and collagen, or by soluble platelet agonists like ADP, thrombin, and thromboxane A2. These adhesive proteins and soluble agonists induce signal transduction via their respective receptors. The various receptor-specific platelet activation signaling pathways converge into common signaling events, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, additional granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. It has become increasingly evident that agonist-induced platelet activation signals also crosstalk with integrin “outside-in” signals to regulate platelet responses. Platelet activation involves a series of rapid positive feedback loops that greatly amplify initial activation signals, and enable robust platelet recruitment and thrombus stabilization. Recent studies have provided novel insight into the molecular mechanisms of these processes. PMID:21071698

  20. 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.

  1. Impaired responsiveness of platelets to epinephrine due to α2A adrenoreceptor deficiency in Male Chinese.

    PubMed

    Lin, Tsun-Mei; Lin, Jih-Shyan; Tseng, Jen-Yu; Wu, Shang-Yin; Chen, Tsai-Yun

    2016-01-01

    Epinephrine is known as a weak, but important, agonist for platelet activation. It has been reported that the responsiveness of platelets to epinephrine was markedly impaired in 6% of Caucasians and in 16% of Japanese. The purpose of this study was to screen and characterize this abnormality in healthy Taiwanese Chinese volunteers. We used aggregometry, flow cytometry and platelet function analyzer (PFA)-100 system to assess in 50 healthy male volunteers the responsiveness of platelets to epinephrine stimulation. Using α2A adrenoceptor antagonist BRL44408 maleate competition and a [(3)H]yohimbin binding assay, we evaluated α2A adrenoceptors on platelets. The aggregation of platelets after stimulation with 10 μM of epinephrine indicated two distinct groups of study participants: 24 (48.0%) good- and 26 (52.0%) impaired-responders to epinephrine. Flow cytometric analysis of platelets after stimulated with 1 μM epinephrine showed that glycoprotein (GP) IIb/IIIa and P-selectin expression of epinephrine good- and impaired-responders were 27.1 ± 11.0% vs. 9.9 ± 5.4% (p = 0.003) and 12.2 ± 6.2% vs. 3.6 ± 3.5% (p < 0.001), respectively. The PFA-100 system showed that epinephrine-impaired-responders had a longer collagen-epinephrine induced closure time. Good-responder platelets incubated with BRL44408 maleate had an impaired response to epinephrine stimulation. [(3)H]yohimbine binding studies showed fewer α2A adrenoreceptors on the platelets of epinephrine-impaired-responders than on those of good-responders. The prevalence of impaired responsiveness to epinephrine was high and probably due to α2A adrenoreceptor deficiency in male Taiwanese Chinese.

  2. Assessment of platelet activation in myeloproliferative disorders with complementary techniques.

    PubMed

    Bermejo, Emilse; Alberto, Maria F; Meschengieser, Susana S; Lazzari, Maria A

    2004-04-01

    Bleeding and thrombosis in myeloproliferative disorders (MPD) are common events, sometimes both are present in the same patient during the course of the disease. Platelet activation in patients with MPD is often suggested. The present study analyses the presence of circulating activated platelets, using simultaneously flow cytometry and aggregometric studies in MPD. We studied 28 patients: 13 with polycythaemia vera, seven with essential thrombocythaemia, and eight chronic myeloid leukaemia. We performed functional tests, aggregation and adenosine triphosphate (ATP) release and flow cytometric assays (mepacrine staining and platelet activation markers CD62, CD63 and fibrinogen binding (B-FG)). Twenty-one MPD samples (75%) had reduced aggregation and ATP release. Acquired delta-SPD was detected in 11 of 28 MPD patients (39%), and we found no association between reduced mepacrine labelling and abnormal ATP release. High levels of activation markers were obtained: CD62 in 19 of 28 patients (68%), CD63 in 13 of 28 patients (46%) and B-FG in 19 of 28 patients (68%). The most prevalent abnormality was a reduced aggregation and ATP release. The lack of association between ATP release and mepacrine labelling suggests that other mechanisms, besides the deficit of intraplatelet ATP/adenosine diphosphate, might occur. High levels of activation markers were also observed. We conclude that both tests are complementary and necessary to understand the functional status of platelets in MPD.

  3. 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.

  4. Platelet associated antibodies

    MedlinePlus

    ... medlineplus.gov/ency/article/003552.htm Platelet-associated antibodies blood test To use the sharing features on ... JavaScript. This blood test shows if you have antibodies against platelets in your blood. Platelets are a ...

  5. 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.

  6. The effect of SK&F 95654, a novel phosphodiesterase inhibitor, on cardiovascular, respiratory and platelet function.

    PubMed Central

    Murray, K. J.; Eden, R. J.; Dolan, J. S.; Grimsditch, D. C.; Stutchbury, C. A.; Patel, B.; Knowles, A.; Worby, A.; Lynham, J. A.; Coates, W. J.

    1992-01-01

    1. SK&F 95654 inhibited the guanosine 3':5'-cyclic monophosphate (cyclic GMP)-inhibited phosphodiesterase (cGI-PDE) with an IC50 value of 0.7 microM. The IC50 values were greater than 100 microM for the other four phosphodiesterase isoenzymes tested. The R-enantiomer of SK&F 95654 (IC50 = 0.35 microM) was a more potent inhibitor of cGI-PDE than was the S-enantiomer (IC50 = 5.3 microM). 2. In the guinea-pig working heart, SK&F 95654 produced a positive inotropic response without altering heart rate. 3. Oral administration of SK&F 95654 to conscious dogs caused dose-dependent increases in left ventricular dp/dtmax in the range 10-50 micrograms kg-1. These positive inotropic responses were maintained for 3 h without simultaneous changes in heart rate or blood pressure. The peak effects on left ventricular dp/dtmax were similar for orally and intravenously administered compound, indicating good oral bioavailability. 4. SK&F 95654 caused a potent inhibition of U46619-induced aggregation in both a human washed platelet suspension (WPS) (IC50 = 70 nM) and in human platelet-rich plasma (PRP) (IC50 = 60 nM), indicating that the compound shows negligible plasma binding. 5. The R-enantiomer of SK&F 95654 was twenty fold more potent as an inhibitor of platelet aggregation than was the S-enantiomer. The similarity of this ratio to that obtained on the cGI-PDE suggests that SK&F 95654 inhibits platelet aggregation via its effects on cGI-PDE. This was also indicated by studies which showed that SK&F 95654 increased adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels and activated cyclic AMP-dependent protein kinase in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1422592

  7. Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

    PubMed Central

    Williams, Caitlin; Bryson-Richardson, Robert J.

    2016-01-01

    Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays. Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction. In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function. PMID:27842370

  8. EXTENDED STORAGE OF BUFFY-COAT PLATELET CONCENTRATES IN PLASMA OR A PLATELET ADDITIVE SOLUTION

    PubMed Central

    Slichter, Sherrill J.; Bolgiano, Doug; Corson, Jill; Jones, Mary Kay; Christoffel, Todd; Bailey, S. Lawrence; Pellham, Esther

    2014-01-01

    Background Platelet concentrates prepared from whole blood in the U.S. are made using the platelet-rich-plasma (PRP) method. The platelet concentrates must be made within 8 hours of blood collection and stored for only 5 days. In Europe and Canada, platelet concentrates are made using the buffy-coat (BC) method from whole blood held overnight at 22°C and storage times may be up to 7 days. Our studies were designed to determine how long BC platelets can be stored in plasma or Plasmalyte while meeting the FDA’s post-storage viability criteria. Study Design, Materials, And Methods Normal subjects donated whole blood that was stored at 22°C for 22 ± 2 hours prior to preparation of BC platelets. Platelets were stored for 5 to 8 days in either plasma or Plasmalyte concentrations of 65% or 80%. Radiolabeled autologous stored versus fresh platelet recoveries and survivals were assessed as well as post-storage in vitro assays. Results BC platelets stored in either plasma or 65% Plasmalyte met FDA post-storage platelet recovery criteria for 7 days but survivals for only 6 days, while storage in 80% Plasmalyte gave very poor results. Both stored platelet recoveries and survivals correlated with the same donor’s fresh results, but the correlation was much stronger between recoveries than survivals. In vitro measures of extent of shape change, morphology score, and pH best predicted post-storage platelet recoveries, while annexin V binding best predicted platelet survivals. Conclusion BC platelets stored in either plasma or 65% Plasmalyte meet FDA’s post-storage viability criteria for 6 days. PMID:24673482

  9. Platelet Function Tests

    MedlinePlus

    PLEASE NOTE: Your web browser does not have JavaScript enabled. Unless you enable Javascript , your ability to navigate and access the features of this website will be limited. ... Proceeds from website advertising help sustain Lab Tests Online. AACC is a not-for-profit organization and does not endorse non-AACC products and services. Advertising & Sponsorship: ...

  10. Congenital platelet function defects

    MedlinePlus

    ... Review Date 2/13/2015 Updated by: Rita Nanda, MD, Assistant Professor of Medicine, Section of Hematology/ ... constitute endorsements of those other sites. Copyright 1997-2017, A.D.A.M., Inc. Duplication for commercial ...

  11. Acquired platelet function defect

    MedlinePlus

    ... Review Date 1/22/2015 Updated by: Rita Nanda, MD, Assistant Professor of Medicine, Section of Hematology/ ... constitute endorsements of those other sites. Copyright 1997-2017, A.D.A.M., Inc. Duplication for commercial ...

  12. Hypothermia and Platelet Dysfunction

    DTIC Science & Technology

    2007-11-02

    cardiopulmonary bypass during cardiac surgery, other major surgery, multiple trauma, cold exposure, and neonatal cold injury.1Ŗ The hemorrhagic diathesis...associated with hypothermic cardiopulmonary bypass during cardiac surgery is considered to be primarily a platelet function defect.I6,17,23 We have...cardiopulmonary bypass during cardiac surgery.,8,24 Consistent with this data, other investigators have recently reported that normothermic cardiopulmonary

  13. Analysis of potassium and calcium imaging to assay the function of opioid receptors.

    PubMed

    Spahn, Viola; Nockemann, Dinah; Machelska, Halina

    2015-01-01

    As the activation of opioid receptors leads to the modulation of potassium and calcium channels, the ion imaging represents an attractive method to analyze the function of the receptors. Here, we describe the imaging of potassium using the FluxOR™ potassium ion channel assay, and of calcium using Fura-2 acetoxymethyl ester. Specifically, we (1) characterize the activation of the G-protein-coupled inwardly rectifying potassium 2 channel by agonists of μ- and δ-opioid receptors with the aid of the FluxOR™ assay in cultured mouse dorsal root ganglion neurons, and (2) describe calcium imaging protocols to measure capsaicin-induced transient receptor potential vanilloid 1 channel activity during opioid withdrawal in transfected human embryonic kidney 293 cells.

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

    SciTech Connect

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

    1987-05-01

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

  15. 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.

  16. Can the laboratory assay of protein C activity assist in monitoring the hemostatic function in pre-eclampsia?

    PubMed

    He, S; Bremme, K; Blombäck, M

    1999-04-01

    . Thus, assays of protein C activity might not always assist in monitoring the hemostatic function during pre-eclamptic pregnancy.

  17. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

    PubMed

    Torto, Trudy A; Li, Shuang; Styer, Allison; Huitema, Edgar; Testa, Antonino; Gow, Neil A R; van West, Pieter; Kamoun, Sophien

    2003-07-01

    Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including

  18. 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.

  19. Blood platelet kinetics and platelet transfusion.

    PubMed

    Aster, Richard H

    2013-11-01

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

  20. 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

  1. 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.

  2. A complementation assay for in vivo protein structure/function analysis in Physcomitrella patens (Funariaceae)

    SciTech Connect

    Scavuzzo-Duggan, Tess R.; Chaves, Arielle M.; Roberts, Alison W.

    2015-07-14

    Here, a method for rapid in vivo functional analysis of engineered proteins was developed using Physcomitrella patens. A complementation assay was designed for testing structure/function relationships in cellulose synthase (CESA) proteins. The components of the assay include (1) construction of test vectors that drive expression of epitope-tagged PpCESA5 carrying engineered mutations, (2) transformation of a ppcesa5 knockout line that fails to produce gametophores with test and control vectors, (3) scoring the stable transformants for gametophore production, (4) statistical analysis comparing complementation rates for test vectors to positive and negative control vectors, and (5) analysis of transgenic protein expression by Western blotting. The assay distinguished mutations that generate fully functional, nonfunctional, and partially functional proteins. In conclusion, compared with existing methods for in vivo testing of protein function, this complementation assay provides a rapid method for investigating protein structure/function relationships in plants.

  3. Abnormal platelet von Willebrand factor (vWF) as a marker of abnormal function in megakaryocytic dysplasia.

    PubMed

    de Cataldo, F; Baudo, F; Redaelli, R; Corno, A R

    1995-03-01

    The myelodysplastic syndromes (MDS) are neoplastic disorders of the hemopoietic system; multilineage involvement is also evidenced by specific cellular dysfunctions. The von Willebrand factor (vWF), synthesized and processed in the megakaryocytes (MK), is stored in the alpha granules of the platelets. The platelet vWF multimeric pattern was studied in 18 patients with MDS, and in 4 with pernicious anemia (PA), to investigate whether the processing of vWF is abnormal in the megakaryocytic dysplasia. An abnormal multimeric pattern was observed in 10/18 MDS and 4/4 PA patients. The abnormality of this specific protein is the discrete expression of the basic disorder, and is reversible when hemopoiesis is normalized. Although the data do not allow any conclusion, abnormal synthesis is the likely explantation of the abnormality.

  4. Platelet mimicry: The emperor's new clothes?

    PubMed

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

    2016-01-01

    Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities to include in vivo targeting of damaged blood vessels and binding to platelet-adhering opportunistic pathogens. We present views for improved, and pharmaceutically viable nanoparticle design strategies.

  5. The blood platelets contribution to innate host defense - what they have learned from their big brothers.

    PubMed

    Zander, Dorit M W; Klinger, Matthias

    2009-06-01

    Bactericidal effects of blood platelets have been known for more than 120 years, but the underlying mechanisms are largely obscure. Keeping in mind structural and functional analogies of platelets to neutrophils, three different mechanisms are thinkable: Engulfment of pathogens, release of microbicidal proteins, and production of reactive oxygen species (ROS). Here, we focus on the release of ROS and a possible contribution of blood plasma and thrombin to the bactericidal effects. Killing of bacteria was evaluated by DNA fluorescence labeling and electron microscopy. Release of ROS by platelets was measured photometrically by cytochrome C and phenol red/peroxidase assays and was further evaluated by topological methods. We found that (i) platelets produce 1500 times less O(2) (-) and 4000 times less H(2)O(2) compared to neutrophils, (ii) ROS do not affect the killing rates, and (iii) no local enrichment of ROS was detectable. On the other hand, thrombin and plasma proteins with a molecular mass of >100 kDa are essential for bactericidal effects. We suggest that platelets contribute to the innate host defense by providing a catalytical surface for synthesis of thrombin. In the presence of a heat-instable plasma protein, thrombin may generate a strong bactericidal complex, which is only effective in close vicinity to the platelet membrane.

  6. 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.

  7. Venous hypertension induces increased platelet reactivity and accumulation in patients with chronic venous insufficiency.

    PubMed

    Lu, Xinwu; Chen, Yujie; Huang, Yin; Li, Weimin; Jiang, Mier

    2006-01-01

    The objective of this study was to determine whether there are changes in platelet activation and rheology in patients with chronic venous insufficiency (CVI) and what their impact is on this disease. Anticoagulated peripheral venous blood collected from 21 patients with CVI and 13 normal control subjects in different bodily positions was incubated either with 0.5 mumol/L adenosine diphosphate (ADP) or without agonist and analyzed by whole blood flow cytometry. Soluble P-selectin was analyzed in obtained sera by enzyme-linked immunosorbent assay. Platelet count was determined by a whole blood analyzer. Circulating platelets were more reactive to stimulation with 0.5 mumol/L ADP in patients with CVI compared with control subjects. There was no statistically significant change in platelet activation without ADP and the level of soluble P-selectin as a function of posture. Under simulated venous hypertension, platelet accumulation was observed in patients with CVI. Patients with CVI had increased platelet reactivity and accumulation during orthostasis, suggesting this might be a contributory factor to CVI pathogenesis.

  8. (Dicer)phering roles of microRNA in platelets.

    PubMed

    Boilard, Eric; Belleannée, Clémence

    2016-04-07

    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.

  9. Bone-to-bone Fixation Enhances Functional Healing of the Porcine Anterior Cruciate Ligament Using a Collagen-Platelet Composite

    PubMed Central

    Murray, Martha M.; Magarian, Elise; Zurakowski, David; Fleming, Braden C.

    2010-01-01

    Purpose The purpose of this study was to determine if providing bony stabilization between the tibia and femur would improve the structural properties of an “enhanced” ACL repair using a collagen-platelet composite when compared to the traditional (Marshall) suture technique. Methods Twelve pigs underwent unilateral ACL transection and were treated with sutures connecting the bony femoral ACL attachment site to the distal ACL stump (LIGAMENT group), or to the tibia via a bone tunnel (TIBIA group). A collagen-platelet composite was placed around the sutures to enhance the biologic repair in both groups. Anteroposterior (AP) knee laxity and the graft structural properties were measured after 15 weeks of healing in both the ACL-repaired and contralateral ACL-intact joints. Results Enhanced ACL repair with bone-to-bone fixation significantly improved yield load and linear stiffness of the ACL repairs (p<0.05) after 15 weeks of healing. However, laxity values of the knees were similar in both groups of repaired knees (p>0.10). Conclusions Using an enhanced ACL suture repair technique that includes bone-to-bone fixation to protect the repair in the initial healing stages resulted in an ACL with improved structural properties after 15 weeks in the porcine model. Clinical Relevance The healing response of an ACL suture repair using a collagen-platelet composite can be enhanced by providing bony stabilization between the tibia and femur to protect the graft during the initial healing process in a translational model. PMID:20810092

  10. Human platelets express authentic CB₁ and CB₂ receptors.

    PubMed

    Catani, M V; Gasperi, V; Catanzaro, G; Baldassarri, S; Bertoni, A; Sinigaglia, F; Avigliano, L; Maccarrone, M

    2010-11-01

    In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB₁) or type-2 (CB₂) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB₁ and CB₂ are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB₁, and to a lesser extent CB₂, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB₁ and CB₂ in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

  11. 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

  12. Protoplast cell death assay to study Magnaporthe oryzae AVR gene function in rice.

    PubMed

    Kanzaki, Hiroyuki; Yoshida, Kentaro; Saitoh, Hiromasa; Tamiru, Muluneh; Terauchi, Ryohei

    2014-01-01

    We describe a protocol for transient gene expression in rice protoplasts and its application to the study of Magnaporthe oryzae avirulence (AVR) gene function. In this assay the gene encoding the firefly luciferase protein is transfected into rice protoplasts by electroporation together with the candidate AVR genes. The luminescence can then be used to assess the viability of rice protoplasts. The hypersensitive response (HR) caused by the interaction between M. oryzae AVR and rice R genes can subsequently be monitored by recording the decrease in luminescence from the transfected cells.

  13. 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.

  14. 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.

  15. Functional characterization of BRCA1 gene variants by mini-gene splicing assay

    PubMed Central

    Steffensen, Ane Y; Dandanell, Mette; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Nielsen, Finn C; Hansen, Thomas vO

    2014-01-01

    Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213−1G>A, c.670+1delG, c.4185+1G>A, and c.5075−1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302−15C>G, c.547+14delG, c.4676−20A>G, c.4987−21G>T, and c.5278−14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members. PMID:24667779

  16. [Renal function and plasma dabigatran level measured at trough by diluted thrombin time assay].

    PubMed

    Martinuzzo, Marta E; Duboscq, Cristina; Viñuales, Estela S; Girardi, Beatriz; Penchasky, Diana; Ceresetto, José; Stemmelin, Germán; Otero, Victoria; Barrera, Luis H; López, Marina S; Otaso, Juan C; Hoyhamburu, José

    2017-01-01

    Dabigatran etexilate (direct thrombin inhibitor) is effective in preventing embolic stroke in patients with atrial fibrillation. It does not require laboratory control, but given the high renal elimination, its measurement in plasma is important in renal failure. The objectives of the study were to verify the analytical quality of the diluted thrombin time assay for measurement of dabigatran plasma concentration (cc), correlate cc with classic coagulation assays, prothrombin time (PT) and activated partial thromboplastin time (APTT), and evaluate them according to the creatinine clearance (CLCr). Forty plasma samples of patients (34 consecutive and 6 suspected of drug accumulation) receiving dabigatran at 150 (n = 19) or 110 (n = 21) mg/12 hours were collected. Blood samples were drawn at 10-14 hours of the last intake. Dabigatran concentration was determined by diluted thrombin time (HemosIl DTI, Instrumentation Laboratory (IL). PT and APTT (IL) were performed on two fotooptical coagulometers, ACL TOP 300 and 500 (IL). DTI presented intra-assay coefficient of variation < 5.4% and inter-assay < 6%, linearity range 0-493 ng/ml. Patients' cc: median 83 (4-945) ng/ml. Individuals with CLCr in the lowest tertile (22.6-46.1 ml/min) showed significantly higher median cc: 308 (49-945), compared to the average 72 (12-190) and highest tertile, 60 (4-118) ng/ml. Correlation between cc and APTT or PT were moderate, r2 = 0.59 and -0.66, p < 0.0001, respectively. DTI test allowed us to quantify plasma dabigatran levels, both in patients with normal or altered renal function, representing a useful tool in clinical situations such as renal failure, pre surgery or emergencies.

  17. Functional characterization of BRCA1 gene variants by mini-gene splicing assay.

    PubMed

    Steffensen, Ane Y; Dandanell, Mette; Jønson, Lars; Ejlertsen, Bent; Gerdes, Anne-Marie; Nielsen, Finn C; Hansen, Thomas vO

    2014-12-01

    Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213-1G>A, c.670+1delG, c.4185+1G>A, and c.5075-1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302-15C>G, c.547+14delG, c.4676-20A>G, c.4987-21G>T, and c.5278-14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members.

  18. Extracts from Trifolium pallidum and Trifolium scabrum aerial parts as modulators of blood platelet adhesion and aggregation.

    PubMed

    Kolodziejczyk-Czepas, Joanna; Olas, Beata; Malinowska, Joanna; Wachowicz, Barbara; Szajwaj, Barbara; Kowalska, Iwona; Oleszek, Wieslaw; Stochmal, Anna

    2013-01-01

    A growing number of reports indicate that some species of clover (Trifolium) may have remarkable medical importance; however, the effects of these plants on blood platelets and hemostasis are inadequately recognized. This work was designed to study the effects of Trifolium pallidum and Trifolium scabrum extracts on the functions of human blood platelets in vitro. Platelet suspensions were preincubated with extracts from aerial parts of T. pallidum (phenolic fraction and clovamide fraction) and T. scabrum (phenolic fraction) at the final concentrations of 12.5, 25, and 50 µg/ml. Then, for platelet activation thrombin (0.1 U/ml), thrombin receptor activating peptide (TRAP; 20 µM), or adenosine diphosphate (ADP; 1 µM) were used. The effects of Trifolium extracts on adhesion of blood platelets to fibrinogen and collagen were determined by enzyme-linked immunosorbent assay (ELISA) method. Platelet aggregation was monitored on a dual-channel Chronolog aggregometer. In these studies, we also compared the action of tested plant extracts with the effects of another antiplatelet plant-derived compound - resveratrol (3,4',5-trihydroxystilbene). The performed assays demonstrated that the tested extracts might influence the platelet functions in vitro. The inhibitory, concentration-dependent effects of all tested extracts on adhesion of thrombin-stimulated platelets to collagen was found. Both extracts from T. pallidum and from T. scabrum reduced the thrombin-induced platelet adhesion to fibrinogen. Furthermore, in the presence of all three extracts, the platelet aggregation induced by thrombin was slightly inhibited. Our results also indicate that the tested plant extracts (at the highest concentrations used of 50 µg/ml), similar to purified resveratrol, inhibit selected steps of platelet activation stimulated by both proteolytic (thrombin) and nonproteolytic agonists (TRAP or ADP). In the comparative studies, T. pallidum and T. scabrum extracts was not found

  19. 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.

  20. Long-term untreated streptozotocin-diabetes leads to increased expression and elevated activity of prostaglandin H2 synthase in blood platelets.

    PubMed

    Siewiera, Karolina; Kassassir, Hassan; Talar, Marcin; Wieteska, Lukasz; Watala, Cezary

    2016-01-01

    In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes.

  1. Human-parathormone assay for use in dogs: validation, sample handling studies, and parathyroid function testing.

    PubMed

    Torrance, A G; Nachreiner, R

    1989-07-01

    Ten commercially available parathormone (PTH) assays were competitively validated, using dilutional parallelism, intra-assay and interassay coefficients of variation, and sensitivity and measured responses of 2 dogs to calcium and EDTA infusions. A 2-site immunoradiometric assay for intact human-PTH was superior to the others for estimating canine-PTH, met the criteria for validity, and was further investigated. A series of sample-handling studies was performed. Serum and plasma samples stored at 24 C lost 15% (n = 5; P less than 0.05) of PTH between 2 and 24 hours. This did not occur at 6 C. The mean PTH concentration of sera from blood samples clotted at 24 C was 6% (P less than 0.05) higher than equivalent EDTA samples. Serum samples stored at 6 and 37 C deteriorated 35% and 100% (n = 5; P less than 0.05), respectively, after 1 week, whereas samples stored at -20 and -70 C for 4 weeks did not deteriorate. There was no significant deterioration of PTH in samples frozen (-40 C) and thawed up to 7 times (n = 5). Parathyroid function testing was investigated by use of 2-hour infusions of disodium EDTA (25 mg/kg/h), 10-minute infusions of calcium gluconate (3 mg of elemental calcium/kg/10 min), and physiologic saline controls (n = 8). Renal function was monitored before and after EDTA infusion by exogenous creatinine clearance. Infusion of disodium EDTA increased mean PTH concentration from 67 (time 0) to 317 and 235 pg/ml at 90 and 180 minutes, respectively (P less than 0.001). Infusion of calcium gluconate decreased mean PTH concentration from 84 (time 0) to 14 and 12 pg/ml at 15 and 60 minutes, respectively (P less than 0.005). There were no observable side effects of the infusions in normal conscious dogs and no differences in exogenous creatinine clearance after EDTA infusion.

  2. Evaluation of four methods for platelet compatibility testing

    SciTech Connect

    McFarland, J.G.; Aster, R.H.

    1987-05-01

    Four platelet compatibility assays were performed on serum and platelet or lymphocyte samples from 38 closely HLA-matched donor/recipient pairs involved in 55 single-donor platelet transfusions. The 22 patients studied were refractory to transfusions of pooled random-donor platelets. Of the four assays (platelet suspension immunofluorescence, PSIFT; /sup 51/Cr release; microlymphocytotoxicity; and a monoclonal anti-IgG assay, MAIA), the MAIA was most predictive of platelet transfusion outcome (predictability, 74% for one-hour posttransfusion platelet recovery and 76% for 24-hour recovery). The only other assay to reach statistical significance was the PSIFT (63% predictability for one-hour posttransfusion recovery). The degree of HLA compatibility between donor and recipient (exact matches v those utilizing cross-reactive associations) was unrelated to the ability of the MAIA to predict transfusion results. The MAIA may be capable of differentiating HLA antibodies, ABO antibodies, and platelet-specific antibodies responsible for failure of HLA-matched and selectively mismatched single-donor platelet transfusions.

  3. 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.

  4. 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)

  5. Proteomics of apheresis platelet supernatants during routine storage: Gender-related differences

    PubMed Central

    Dzieciatkowska, Monika; D‘Alessandroa, Angelo; Burke, Timothy A.; Kelher, Marguerite R.; Moore, Ernest E.; Banerjee, Anirban; Silliman, Christopher C.; West, Bernadette F.; Hansen, Kirk C.

    2015-01-01

    Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernat