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Sample records for platelet aggregation inhibitor

  1. Platelet aggregation Inhibitors from Hametophagous Animals

    PubMed Central

    Francischetti, Ivo M. B.

    2010-01-01

    Salivary glands from blood-sucking animals (e.g., mosquitoes, bugs, sandflies, fleas, ticks, leeches, hookworms, bats) are a rich source of bioactive molecules that counteract hemostasis in a redundant and synergistic manner. This review discusses recent progress in the identification of salivary inhibitors of platelet aggregation, their molecular characterization, and detailed mechanism of action. Diversity of inhibitors is remarkable, with distinct families of proteins characterized as apyrases that enzymatically degrade ADP or as collagen-binding proteins that prevent its interaction with vWF, or platelet integrin α2β1 or GPVI. Molecules that bind ADP, TXA2, epinephrine, or serotonin with high affinity have also been cloned, expressed, and their structure determined. In addition, a repertoire of antithrombins and an increasingly number of RGD and non-RGD disintegrins targeting platelet αIIbβ3 have been reported. Moreover, metalloproteases with fibrinogen(olytic) activity and PAF phosphorylcholine hydrolase are enzymes that have been recruited to the salivary gland to block platelet aggregation. Platelet inhibitory prostaglandins, lysophosphatydilcholine, adenosine, and nitric oxide (NO)-carrying proteins are other notable examples of molecules from hematophagous salivary secretions (herein named sialogenins) with antihemostatic properties. Sialogenins have been employed as tools in biochemistry and cell biology and also display potential therapeutic applications. PMID:20035779

  2. Short-term exposure of platelets to glucose impairs inhibition of platelet aggregation by cyclooxygenase inhibitors.

    PubMed

    Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

    2011-01-01

    Aspirin treatment reduces cardiovascular events and deaths in high-risk non-diabetic patients, but not in patients suffering from diabetes. In these patients, hyperglycemia has been found to cause reduced platelet sensitivity to aspirin. It is supposed that long-term exposure of platelets to glucose leads to non-enzymatic glycosylation and impairs aspirin inhibition of platelet aggregation. On the other hand, short-term exposure of platelets to glucose also attenuates the effect of aspirin on platelets. The aim of the present work was to analyse the effect of short-term exposure of glucose on the inhibition of platelet aggregation by aspirin and other cyclooxygenase (COX) inhibitors. Already a 15 min exposure of platelets to glucose impaired aspirin inhibition of the platelet aggregation induced by collagen, thrombin, adenosine diphosphate (ADP), and arachidonic acid (AA). Aspirin inhibition of platelet aggregation in platelet-rich plasma (PRP) was attenuated by 5.6, 11.2, 16.8, and 22.4 mM of glucose in a concentration-dependent way. The same effect was observed with indomethacin and acetaminophen used as cyclooxygenase inhibitors instead of aspirin. N-methyl-L-arginine, an inhibitor of nitric oxide synthase, prevented the effect of glucose on aspirin, indomethacin and acetaminophen inhibition of platelet aggregation. Other monosaccharides, for example fructose and galactose, impaired aspirin inhibition as did glucose. Lactic acid (0.1, 0.2, 0.4, 0.8 mM), the end product of anaerobic glycolysis in platelets, impaired the inhibition of platelet aggregation with aspirin in a concentration-dependent way but did not affect indomethacin. It is suggested that lactic acid might be a mediator of the effect of glucose on aspirin inhibition in platelets.

  3. New platelet aggregation inhibitors based on pyridazinone moiety.

    PubMed

    Costas, Tamara; Costas-Lago, María Carmen; Vila, Noemí; Besada, Pedro; Cano, Ernesto; Terán, Carmen

    2015-04-13

    New series of pyridazinone derivatives (4, 5 and 6) were synthesized in good yields following a synthetic strategy based on singlet oxygen oxidation of alkyl furans, in which a suitable β(α)-substituted γ-hydroxybutenolide (10 or 11) or a bicyclic lactone (12 or 13) was the key intermediate. The synthesized compounds were tested in vitro as antiplatelet agents and some of them (compounds 4b, 4d and 5b) exhibited potent inhibitory effects on collagen-induced platelet aggregation with IC50 values in the low μM range. Studies performed with the most active compound of these series (4b) demonstrated its lack of activity as inhibitor of platelet aggregation induced by other agonists as thrombin, ionomycin or U-46619 suggesting a selective action on the biochemical mechanisms triggered by collagen in the platelets.

  4. Substituted pyrazolylhydrazones: a new class of platelet aggregation inhibitors.

    PubMed

    Silveira, I A; Paulo, L G; Miranda, A L; Rocha, S O; Freitas, A C; Barreiro, E J

    1991-01-01

    A series of 5-pyrazolylhydrazone derivatives (I) were designed to be mixed hybrid isosteres of both BW-755C and CBS-1108 which belong to the class of dual cyclooxygenase and 5-lipoxygenase inhibitors. Pharmacological evaluation of some members of this series (Ia, 1-formyl-3,4-methylenedioxy-6-nitrobenzene-5-(1-phenyl-3-methyl-4-nit ropyrazolyl)hydrazone; Ib, 2-formylfurane-5-(1-phenyl-3-methyl-4-nitropyrazolyl)hydr azo ne;Ic, (E)-2-(formylethenylfurane)-5-(1-phenyl-3-methyl-4-nitropyrazol yl)hydrazone showed that they inhibit the in vitro platelet aggregation of citrated platelet-rich rabbit plasma induced by ADP (5 microM), collagen (5 micrograms/ml) and arachidonic acid (100 microM). Compounds Ia and Ic at 100 microM concentration showed 49% and 58% inhibition, respectively, of ADP-induced aggregation. In the arachidonic acid-induced aggregation, compounds Ia and Ib at 100 microM concentration fully inhibited platelet aggregation. All compounds significantly inhibited the collagen-induced aggregation. In contrast, indomethacin (10 microM) showed 100% and 85% aggregation inhibition against arachidonic acid and collagen, respectively, and was inactive in the ADP-induced aggregation test. These results suggest that the structure-activity relationship in this series of compounds is dependent on the hydrazone moiety at position 5 of the pyrazole ring and on the distance between the aryl ring and the pyrazole ring and that the 2-furyl ring is at the optimal distance for the maximal activity.

  5. New pyrazolylhydrazone derivatives as inhibitors of platelet aggregation.

    PubMed

    da Silveira, I A; Paulo, L G; de Miranda, A L; Rocha, S O; Freitas, A C; Barreiro, E J

    1993-07-01

    A series of 5-pyrazolylhydrazone derivatives was designed to be mixed hybrid isosteres of both BW755C and CBS-1108, which belong to the class of dual cyclo-oxygenase and 5-lipoxygenase inhibitors. Some derivatives of this series inhibit the in-vitro platelet aggregation of citrated platelet-rich rabbit plasma induced by ADP (5 microM), collagen (5 micrograms mL-1) and arachidonic acid (100 microM). The structure-activity relationships of this class of compounds were determined from these results. When ADP is used as the aggregation inducer, the presence of free oxygenated substituents at the p-position in the phenyl subunit of the hydrazone moiety favours inhibitory activity; p-methoxyformylbenzene-5-(1-phenyl-3-methyl-4-nitropyrazolyl )hydrazone (100 microM), which has a methoxy group at this position was the most active with 62.8% inhibition of aggregation. In contrast, substitution in the aryl ring does not affect the aggregation induced by collagen, whereas the non-substituted compound, formylbenzene-5-(1-phenyl-3-methyl-4-nitropyrazolyl)hydra zon e, showed similar activity to those of substituted derivatives. In the arachidonic acid assays, the presence of an aryl ring linked to the hydrazone moiety, with an adequate electronic density at the ring due to the nature of its substituents, is an important structural requirement for inhibitory activity.

  6. Human platelet aggregation inhibitors from thyme (Thymus vulgaris L.).

    PubMed

    Okazaki, Kenji; Kawazoe, Kazuyoshi; Takaishi, Yoshihisa

    2002-06-01

    Two antiaggregant compounds, thymol (compound 1) and 3,4,3',4'-tetrahydroxy-5,5'-diisopropyl-2,2'-dimethylbiphenyl (compound 2) were isolated from the leaves of thyme (Thymus vulgaris L.). The structures were determined by (1)H-, (13)C-NMR and mass spectra (MS) studies. These compounds inhibited platelet aggregation induced by collagen, ADP, arachidonic acid (AA) and thrombin except that compound 2 did not inhibit platelet aggregation induced by thrombin.

  7. Cardamom extract as inhibitor of human platelet aggregation.

    PubMed

    Suneetha, W Jessie; Krishnakantha, T P

    2005-05-01

    The inhibitory activity of cardamom extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes, respectively, obtained from blood of healthy volunteers. Human platelets were subjected to stimulation with a variety of agonists including ADP (2.5 mM), epinephrine (2.5 mM), collagen (10 mM), calcium ionophore A 23187 (6 microM) and ristocetin (1.25 microg/mL). The IC50 were 0.49, 0.21, 0.55 and 0.59 mg with ADP, epinephrine, collagen and calcium ionophore A 23187, respectively, and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.14 and 0.70 mg and time dependent at IC50. Lipid peroxidation induced by iron--ascorbic acid system in platelet membranes was analysed with malondialdehyde (MDA) as an index. An increase in concentration of cardamom has decreased the MDA formation significantly. Hence, it may be said that aqueous extract of cardamom may have component(s), which protect platelets from aggregation and lipid peroxidation.

  8. Dabigatran and rivaroxaban do not affect AA- and ADP-induced platelet aggregation in patients receiving concomitant platelet inhibitors.

    PubMed

    Olivier, Christoph B; Weik, Patrick; Meyer, Melanie; Weber, Susanne; Diehl, Philipp; Bode, Christoph; Moser, Martin; Zhou, Qian

    2016-08-01

    Dabigatran and rivaroxaban are novel, vitamin K-independent oral anticoagulants (NOACs) and act via antagonism of the coagulation factor (F) IIa (dabigatran) or FXa (rivaroxaban), respectively. Compared to vitamin-K-antagonists, NOACs have shown non-inferiority of risk and benefit in patients with non valvular atrial fibrillation (AF). In clinical practice there is increasing use of NOACs combined with platelet inhibitors in patients with AF and coronary artery disease. However, whether NOACs affect the function of platelet inhibitors remains incompletely known. This observational study aimed to assess the platelet function in patients receiving dabigatran or rivaroxaban and concomitant platelet inhibitors. A single centre observational study was performed analysing the platelet aggregation of patients treated with dabigatran or rivaroxaban with or without concomitant platelet inhibitors. Measurements before the initiation of NOAC therapy served as the respective control group. Platelet aggregation was measured by multiple electrode aggregometry and was induced with adenosine diphosphate (ADP, 6.5 µM) and arachidonic acid (AA, 0.5 mM), respectively. In order to evaluate whether NOACs interact with platelet inhibition by ASA or the P2Y12-antagonist clopidogrel, 87 patients were grouped according to their concomitant antiplatelet medication. Comparing the ADP- and AA-induced platelet aggregation in patients without concomitant platelet inhibitors (n = 45) no significant differences under therapy with dabigatran (d) or rivaroxaban (r) compared to the control group (c) were observed. In patients taking clopidogrel as a concomitant platelet inhibitor (n = 21), neither dabigatran nor rivaroxaban affected the ADP-induced platelet aggregation (c 20 ± 11, d 21 ± 14, r 18 ± 8 AU*min, p = 0.200). Patients receiving dabigatran or rivaroxaban in combination with ASA (n = 42; 21 ASA only, 21 ASA + clopidogrel) showed no significant differences of the AA

  9. Spice active principles as the inhibitors of human platelet aggregation and thromboxane biosynthesis.

    PubMed

    Raghavendra, R H; Naidu, K Akhilender

    2009-07-01

    Spice active principles are reported to have anti-diabetic, anti-hypercholesterolemic, antilithogenic, anti-inflammatory, anti-microbial and anti-cancer properties. In our previous report we have shown that spices and their active principles inhibit 5-lipoxygenase and also formation of leukotriene C4. In this study, we report the modulatory effect of spice active principles viz., eugenol, capsaicin, piperine, quercetin, curcumin, cinnamaldehyde and allyl sulphide on in vitro human platelet aggregation. We have demonstrated that spice active principles inhibit platelet aggregation induced by different agonists, namely ADP (50microM), collagen (500microg/ml), arachidonic acid (AA) (1.0mM) and calcium ionophore A-23187 (20microM). Spice active principles showed preferential inhibition of arachidonic acid-induced platelet aggregation compared to other agonists. Among the spice active principles tested, eugenol and capsaicin are found to be most potent inhibitors of AA-induced platelet aggregation with IC50 values of 0.5 and 14.6microM, respectively. The order of potency of spice principles in inhibiting AA-induced platelet aggregation is eugenol>capsaicin>curcumin>cinnamaldehyde>piperine>allyl sulphide>quercetin. Eugenol is found to be 29-fold more potent than aspirin in inhibiting AA-induced human platelet aggregation. Eugenol and capsaicin inhibited thromboxane B2 (TXB2) formation in platelets in a dose-dependent manner challenged with AA apparently by the inhibition of the cyclooxygenase (COX-1). Eugenol-mediated inhibition of platelet aggregation is further confirmed by dose-dependent decrease in malondialdehyde (MDA) in platelets. Further, eugenol and capsaicin inhibited platelet aggregation induced by agonists-collagen, ADP and calcium ionophore but to a lesser degree compared to AA. These results clearly suggest that spice principles have beneficial effects in modulating human platelet aggregation.

  10. Use of selective-serotonin reuptake inhibitors and platelet aggregation inhibitors among individuals with co-occurring atherosclerotic cardiovascular disease and depression or anxiety

    PubMed Central

    Thornton, J Douglas; Agarwal, Parul; Sambamoorthi, Usha

    2016-01-01

    Objective: Medications commonly used to treat heart disease, anxiety, and depression can interact resulting in an increased risk of bleeding, warranting a cautious approach in medical decision making. This retrospective, descriptive study examined the prevalence and the factors associated with the use of both selective-serotonin reuptake inhibitor and platelet aggregation inhibitor among individuals with co-occurring atherosclerotic cardiovascular disease and anxiety or depression. Methods: Respondents aged 22 years and older, alive throughout the study period, and diagnosed with co-occurring atherosclerotic cardiovascular disease and anxiety or depression (n = 1507) in years 2007 through 2013 of the Medical Expenditures Panel Survey were included. The use of treatment was grouped as follows: selective-serotonin reuptake inhibitor and platelet aggregation inhibitor, selective-serotonin reuptake inhibitor or platelet aggregation inhibitor, and neither selective-serotonin reuptake inhibitor nor platelet aggregation inhibitor. Results: Overall, 16.5% used both selective-serotonin reuptake inhibitor and platelet aggregation inhibitor, 61.2% used selective-serotonin reuptake inhibitor or platelet aggregation inhibitor, and 22.3% used neither selective-serotonin reuptake inhibitor nor platelet aggregation inhibitor. Respondents aged over 65 years (adjusted odds ratio = 1.93 (95% confidence interval = 1.08–3.45)) and having a diagnosis of diabetes (adjusted odds ratio = 1.63 (95% confidence interval = 1.15–2.31)) and hypertension (adjusted odds ratio = 1.84 (95% confidence interval = 1.04–3.27)) were more likely to be prescribed the combination. Conclusion: The drug interaction was prevalent in patients who are already at higher risk of health disparities and worse outcomes thus requiring vigilant evaluation. PMID:28348738

  11. Substituted alpah-methylbenzyl and tricyclic arylalkyl lactamimides as inhibitors of blood platelet aggregation.

    PubMed

    Grisar, J M; Claxton, G P; MacKenzie, R D

    1976-04-01

    N-[1-(p-Phenoxyphenyl)ethyl]hexahydro-2H-azepin-2-imine hydrochloride (10) and N-[1-(2-dibenzothienyl)-ethyl]hexahydro-2H-azepin-2-imine hydrochloride (22) were found to inhibit in vitro aggregation of human blood platelets induced by ADP with minimal release of procoagulant platelet factor 3. The compounds were selected from a series of substituted alpha-methylbenzyl and tricyclic arylalkyl lactamimides that were free of hypoglycemic and diuretic effects. Compounds 10 and 22, as well as N-[1-(1-naphthyl)ethyl]hexahydro-2H-azepin-2-imine hydrochloride (I) and N-(2,2-diphenylpentyl)hexahydro-2H-azepin-2-imine hydrochloride (II), were evaluated for effects on ADP-induced platelet aggregation after repeated oral administration to guinea pigs. Compound II (RMI 12,366A) showed in vivo activity in this system 2 h after the last of four daily doses of 100 mg/kg po.

  12. Acetal phosphatidic acids: novel platelet aggregating agents.

    PubMed

    Brammer, J P; Maguire, M H; Walaszek, E J; Wiley, R A

    1983-05-01

    1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids

  13. C-phycocyanin, a very potent and novel platelet aggregation inhibitor from Spirulina platensis.

    PubMed

    Hsiao, George; Chou, Po-Hsiu; Shen, Ming-Yi; Chou, Duen-Suey; Lin, Chien-Huang; Sheu, Joen-Rong

    2005-10-05

    The aim of this study was to systematically examine the inhibitory mechanisms of C-phycocyanin (C-PC), one of the major phycobiliproteins of Spirulina platensis (a blue-green alga), in platelet activation. In this study, C-PC concentration-dependently (0.5-10 nM) inhibited platelet aggregation stimulated by agonists. C-PC (4 and 8 nM) inhibited intracellular Ca2+ mobilization and thromboxane A2 formation but not phosphoinositide breakdown stimulated by collagen (1 microg/mL) in human platelets. In addition, C-PC (4 and 8 nM) markedly increased levels of cyclic GMP and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser(157) phosphorylation. Rapid phosphorylation of a platelet protein of Mw 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by C-PC (4 and 8 nM). In addition, C-PC (4 and 8 nM) markedly reduced the electron spin resonance (ESR) signal intensity of hydroxyl radicals in collagen (1 microg/mL)-activated platelets. The present study reports on a novel and very potent (in nanomolar concentrations) antiplatelet agent, C-PC, which is involved in the following inhibitory pathways: (1) C-phycocyanin increases cyclic GMP/VASP Ser157 phosphorylation and subsequently inhibits protein kinase C activity, resulting in inhibition of both P47 phosphorylation and intracellular Ca2+ mobilization, and (2) C-PC may inhibit free radicals (such as hydroxyl radicals) released from activated platelets, which ultimately inhibits platelet aggregation. These results strongly indicate that C-PC appears to represent a novel and potential antiplatelet agent for treatment of arterial thromboembolism.

  14. ACE and platelet aggregation inhibitors from Tamarix hohenackeri Bunge (host plant of Herba Cistanches) growing in Xinjiang

    PubMed Central

    Xing, Yachao; Liao, Jing; Tang, Yingzhan; Zhang, Peng; Tan, Chengyu; Ni, Hui; Wu, Xueqin; Li, Ning; Jia, Xiaoguang

    2014-01-01

    Background: Tamarix hohenackeri Bunge is a salt cedar that grows widespread in the desert mountains in Xinjiang. T. hohenackeri has not been investigated earlier, although there are many reports of phytochemical work on other Tamarix species. Materials and Methods: To find out natural angiotensin-converting enzyme (ACE) inhibitor and platelet aggregation inhibitors, the bioactive extract (ethyl acetate [EtOAc] fraction) from the dried aerial parts of T. hohenackeri were investigated. The active fraction was purified by repeated column chromatography, including silica gel, Sephadex LH-20 column, medium-pressure liquid chromatography (MPLC) (polyamide column) and high-performance liquid chromatography (HPLC). The isolated major constituents were tested for their anti-platelet aggregation activity. Results: Bioassay-directed separation of the EtOAc fraction of the 70% ethanol extract from the air-dried aerial parts of T. hohenackeri led to the isolation of a new triterpenoid lactone (1), together with 13 known compounds (2-14). It was the first time to focus on screening bioactive constituents for this plant. The chemical structures were established on the basis of spectral data (ESI-MS and NMR). The results showed that the flavonoid compounds (7 and 8) and phenolic compounds (9, 10, 11, and 14) were potential ACE inhibitors. And the flavonoid compounds (5 and 7) showed significant anti-platelet aggregation activities. Conclusion: On the basis of the chemical and biological data, the material basis of ACE inhibitory activity for the active part was the phenolic constituents. However, the flavonoid compounds were responsible for the anti-platelet aggregation. The primary structure and activity relationship were also discussed respectively. PMID:24914275

  15. Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom.

    PubMed

    Saoud, Samah; Chérifi, Fatah; Benhassine, Traki; Laraba-Djebari, Fatima

    2016-12-07

    The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni(2+) and Mg(2+) completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy.

  16. Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

    PubMed Central

    Polverino de Laureto, P.; Scaramella, E.; De Filippis, V.; Marin, O.; Doni, M. G.; Fontana, A.

    1998-01-01

    Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal

  17. [Platelet aggregation inhibitors and vitamin K antagonists in stomatology and maxillo-facial surgery].

    PubMed

    Lévesque, H; Péron, J-M

    2003-04-01

    Anti-thrombin, anti-platelet and anti-vitamin K agents are common treatments given for the prevention of thrombotic or embolic events. In oral or maxillofacial surgery, the question is whether oral anticoagulants or anti-platelets should be discontinued or not: temporary interruption could limit the risk of bleeding, but would increase the risk of thrombosis, and inversely. The best risk/benefit ratio must be achieved, but in many cases anti-platelet agents or even anti-vitamin K agents can be continued. In case of "potentially hemorrhagic" oral surgery, anti-platelet agents should be interrupted about ten days before the intervention. Anti-vitamin K agents should be interrupted four days before surgery and if required, replaced by low-molecular weight heparin at preventive or curative doses. In the very large majority of patients however, surgery (avulsion for example) can be performed without interrupting anticoagulation. Data in the literature have demonstrated that despite the theoretical risk of hemorrhage, bleeding is minimal and can be easily controlled by local treatments; the risk of thrombotic or thromboembolic events after discontinuing anticoagulation is much greater. A close collaboration between the dentist, the stomatologist, and the clinician is required to determine whether anti-thrombosis prevention should be continued and whether the anticoagulation level is adapted to the thrombotic risk. Good surgical technique and local anti-hemorrhagic treatment are essential in oral surgery, particularly in patients taking anti-platelets or oral anticoagulants.

  18. [Efficacy of clopidogrel as ADP-dependent platelet aggregation inhibitor. Study on individuals with coronary artery disease].

    PubMed

    Izaguirre Avila, R; de la Peña, A; González Pacheco, H; Ramírez Gutiérrez, A; González Valdez, H; Quiroz, A; Cortina, E; Huerta, M; Lupi, E

    2000-01-01

    Acetyl-salicylic acid inhibits thromboxane A2 production and reduces the risk of vascular occlusive events by 20 to 25%. Ticlopidine inhibits ADP-dependent platelet aggregation and reduces the same risk by 30 to 35%, but produces some adverse effects. Clopidogrel is a ticlopidin-derived antiplatelet-drug, with the same mechanism of action; reduces the expression of the glycoprotein IIb/IIIa, the fibrinogen receptor on the platelet surface. Clopidogrel has the same clinical efficacy of ticlopidin and lowers the incidence of adverse effects. In this study, we evaluated the effects of one daily dosis of 75 mg of clopidogrel on platelet function in 33 subjects with coronary artery disease. Before treatment and after the 6th and 12th week, the following parameters were evaluated: 5 microM-ADP and 20 micrograms/mL collagen-induced platelet aggregation, bleeding time and fibrinogen concentration. In basal and in the 6th and 12th week samples, ADP-induced platelet aggregation was 90.7% +/- 13.2, 54.6% +/- 23.2 and 49.2% +/- 23.7 respectively, that represents a significant reduction of 38.6% and 44.4%. Reduction of collagen-induced platelet aggregation was not significative. Plasmatic fibrinogen did not suffer variation during treatment. Bleeding time was significant prolonged from 4.1 minutes to 15.4 and 14.6 minutes (3.7-3.5 times compared with the test before treatment). There were no haemorrhagic complications, only digestive discomfort in fewer than 3% of patients. We concluded that clopidogrel is a safe and efficacious drug for patients, it efficiently reduces ADP-induced platelet aggregation and prolongs bleeding time.

  19. The Heparin-Induced Thrombocytopenia and Thrombosis Syndrome: Treatment with Intraarterial Urokinase and Systemic Platelet Aggregation Inhibitors

    SciTech Connect

    Murphy, Kenneth D.; McCrohan, Gerard; DeMarta, Deborah A.; Shirodkar, Nitin B.; Kwon, Oun J.; Chopra, Paramjit S.

    1996-03-15

    We report a case of the heparin-induced thrombocytopenia and thrombosis syndrome presenting with acute ischemia of a lower limb. The patient was successfully treated by withdrawal of heparin products, intraarterial urokinase, and platelet anti-aggregation therapy consisting of Dextran and aspirin.

  20. Platelet aggregation test

    MedlinePlus

    ... disorders Uremia (a result of kidney failure ) Von Willebrand disease (a bleeding disorder) Risks There is very little ... vasculitis Platelet count Polycythemia vera Prerenal azotemia Von Willebrand disease Review Date 1/27/2015 Updated by: Yi- ...

  1. 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

  2. Blood platelet aggregation and personality traits.

    PubMed

    Jenkins, C D; Thomas, G; Olewine, D; Zyzanski, S J; Simpson, M T; Hames, C G

    1975-12-01

    Changes in blood platelet aggregation may precipitate episodes of arterial occlusive diseases. Little is known, however, regarding the influence of psychological traits, emotional states and other behavioral stressors on platelet aggregation phenomena. This study examined 46 healthy college men at rest and after submaximal treadmill exercise. Associations were found between the duration of platelet aggregation and a number of scores from the California Psychological Inventory and self-administered anxiety scales. The more socially adequate, poised and dominant persons--those with more mature ego development and less overt anxiety--had platelets with more prolonged aggregation reactions to the in vitro introduction of noradrenalin. Irreversible aggregation of platelets occurred more regularly to lower in vitro concentrations of noradrenalin in platelet samples drawn from subjects who were less anxious and tended to be more rigidly defensive. It is premature to attempt to derive clinical implications from this exploratory work, but some implications for the design of future research are discussed.

  3. Labile aggregation stimulating substance, free fatty acids, and platelet aggregation.

    PubMed

    Gerrard, J M; White, J G; Krivit, W

    1976-01-01

    Labile aggregation stimulating substance (LASS), an intermediate produced during platelet biosynthesis of PGE2 and PGF2alpha, acts as a physiologic intercellular messenger to promote platelet aggregation and the release reaction. The activity is formed by intact cells after physiologic stimulation or can be generated from platelet membrane fractions after combination with arachidonate. In the present investigation, small amounts of polyunsaturated fatty acids added to an incubation mixture of platelet microsomes and arachidonate were found to significantly inhibit subsequent platelet aggregation. Saturated and mono-unsaturated fatty acids in the same concentrations were without effect. However, in higher concentrations mono-unsaturated fatty acids were found to be inhibitory and stearic acid was found to enhance subsequent platelet aggregation. The inhibition caused by the polyunsaturated fatty acid, linoleate, was shown to be the result of an effect on the production of LASS through an interaction with the platelet enzyme responsible for conversion of arachidonate to LASS. In contrast, stearic acid was found to enhance platelet aggregation by acting on the platelets and not directly on LASS production. The results suggest that small changes in the fatty acid composition of platelet phospholipids could significantly influence platelet reactivity.

  4. Sequential sup 1 H NMR assignments of kistrin, a potent platelet aggregation inhibitor and glycoprotein IIb-IIIa antagonist

    SciTech Connect

    Adler, M.; Wagner, G. )

    1992-02-04

    Sequence-specific nuclear magnetic resonances assignments have been obtained for the protons of kistrin. Kistrin is a small naturally occurring snake venom protein that inhibits platelet aggregation by blocking the interaction of fibrinogen with the membrane-bound glycoprotein IIb-IIIa (GP IIb-IIIa), a receptor from the integrin family. Kistrin has an Arg-Gly-Asp sequence which is believed to form an adhesion recognition sequence that is essential for activity. Therefore, the interaction between kistrin and GP IIb-IIIa may provide important information on the motif used by integrins to recognize their target proteins which bear RGD sequences. Kistrin consists of 68 residues and contains six intramolecular disulfide bonds. Although one-third of the amide protons are protected from exchange with the solvent, there appears to be little or no regular secondary structure. The large number of NOE's between residues separated by two and three positions in the sequence indicates that the protein contains a large number of tightly packed loops. Along with the sequential assignments, this paper also discusses the construction and use of computerized data bases for manipulating NMR results. A strategy for computer-assisted sequential resonance using these data bases is also presented.

  5. The influence of erythrocyte aggregation on induced platelet aggregation.

    PubMed

    Ott, C; Lardi, E; Schulzki, T; Reinhart, W H

    2010-01-01

    Red blood cells (RBCs) affect platelet aggregation in flowing blood (primary hemostasis). We tested the hypothesis that RBC aggregation could influence platelet aggregation. RBC aggregation was altered in vitro by: (i) changing plasma aggregatory properties with 3.7 g% dextran 40 (D40), 3.0 g% dextran 70 (D70) or 1.55 g% dextran 500 (D500); (ii) changing RBC aggregatory properties by incubating RBCs in 50 mU/ml neuraminidase for 60 min (reduction of the surface sialic acid content, thus reducing electrostatic repulsion) and subsequent RBC resuspension in platelet rich plasma (PRP) containing 1 g% dextran 70. RBC aggregation was assessed with the sedimentation rate (ESR). Platelet aggregation was measured: (i) in flowing whole blood with a platelet function analyzer PFA-100(R), which simulates in vivo conditions with RBCs flowing in the center and platelets along the wall, where they adhere to collagen and aggregate; and (ii) in a Chrono-log 700 Aggregometer, which measures changes of impedance by platelet aggregation in whole blood or changes in light transmission in PRP. We found that RBC aggregation increased with increasing molecular weight of dextran (ESR: 4 +/- 3 mm/h, 34 +/- 14 mm/h and 89 +/- 23 mm/hfor D40, D70 and D500, respectively, p < 0.0001) and with neuraminidase-treated RBCs (76 +/- 27 mm/h vs 27 +/- 8 mm/h, respectively, p < 0.0001). Platelet aggregation measured in whole blood under flow conditions (PFA-100) and without flow (Chronolog Aggregometer) was not affected by RBC aggregation. Our data suggest that RBC aggregation does not affect platelet aggregation in vitro and plays no role in primary hemostasis.

  6. 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.

  7. Geldanamycin disrupts platelet-membrane structure, leading to membrane permeabilization and inhibition of platelet aggregation.

    PubMed Central

    Suttitanamongkol, S; Gear, A R; Polanowska-Grabowska, R

    2000-01-01

    Geldanamycin (GA), a benzoquinoid ansamycin antibiotic, has been used as a tyrosine kinase inhibitor and an anti-tumour agent and is known to bind to heat-shock protein 90. In the present study on human platelets we have found that GA inhibited platelet aggregation induced by ADP, thrombin and the thrombin-receptor-activating peptide and caused platelet plasma-membrane damage, detected by leakage of adenine nucleotides as well as serotonin. Scanning electron microscopy (SEM) revealed that platelet exposure to GA led to the formation of holes or fenestrations in the platelet plasma membrane, confirming GA's ability to initiate membrane damage. In addition, GA itself caused both the dephosphorylation and phosphorylation of proteins in resting platelets and prevented agonist-induced phosphorylation of pleckstrin, the 20-kDa myosin light chain and other proteins. Another ansamycin, herbimycin A, also inhibited platelet aggregation, but caused minimal membrane permeabilization, as detected by (3)H release from platelets labelled previously with [(3)H]adenine, and much less membrane damage, revealed by SEM. Overall, GA is able to disrupt membrane structure and inhibit platelet aggregation, an ability which may be linked to alterations in the activity of protein kinases and phosphatases. PMID:10620508

  8. In vitro model of platelet aggregation in stenotic arteries

    SciTech Connect

    Morley, D.; Santamore, W.P.

    1988-07-01

    Clinical and experimental evidence suggest a strong relationship between arterial stenosis, platelet aggregation, and subsequent thrombus formation. To facilitate the study of platelet accumulation in stenotic arteries, we developed an in vitro preparation. Arterial segments were perfused with whole citrated blood. A stenosis was created by applying an external plastic constrictor to the artery. Platelet accumulation within the stenosis was assessed by scanning electron microscopy and by radioactive counts from Indium-111 labeled platelets. Utilizing this preparation, 30 carotid arterial segments from 10 mongrel dogs were perfused at 100 mmHg for 15 min. In 10 arteries without a stenosis, scanning electron microscopy and radioactive counts demonstrated little platelet accumulation. In contrast, extensive platelet aggregation was observed in 10 arteries with stenoses. Moreover, in 10 stenotic arteries exposed to the thromboxane mimetic, U46619 (Upjohn Diagnostic Group), scanning electron microscopy and radioactive counts demonstrated a significant increase in platelet deposition. Conversely, we demonstrated a dimunition of platelet accumulation in stenosed arterial segments exposed to the prostacyclin analogue platelet inhibitor, Iloprost. The in vitro preparation allows precise control of hemodynamic variables and makes it possible to perform multiple tests on segments of the same vessel from the same animal.

  9. Acid soluble, pepsin resistant platelet aggregating material

    SciTech Connect

    Schneider, M.D.

    1982-08-31

    Disclosed is an acid soluble, pepsin resistant, platelet aggregating material isolated from equine arterial tissue by extraction with dilute aqueous acid. The method of isolation and use to control bleeding are described. 4 figs.

  10. Simplagrin, a Platelet Aggregation Inhibitor from Simulium nigrimanum Salivary Glands Specifically Binds to the Von Willebrand Factor Receptor in Collagen and Inhibits Carotid Thrombus Formation In Vivo

    PubMed Central

    Chagas, Andrezza C.; McPhie, Peter; San, Hong; Narum, David; Reiter, Karine; Tokomasu, Fuyuki; Brayner, Fabio A.; Alves, Luiz C.; Ribeiro, José M. C.; Calvo, Eric

    2014-01-01

    Background Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin. Methods and Findings Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography. Light-scattering studies showed that Simplagrin has an elongated monomeric form with a hydrodynamic radius of 5.6 nm. Simplagrin binds to collagen (type I-VI) with high affinity (2–15 nM), and this interaction does not involve any significant conformational change as determined by circular dichroism spectroscopy. Simplagrin-collagen interaction is both entropically and enthalpically driven with a large negative ΔG, indicating that this interaction is favorable and occurs spontaneously. Simplagrin specifically inhibits von Willebrand factor interaction with collagen type III and completely blocks platelet adhesion to collagen under flow conditions at high shear rates; however, Simplagrin failed to block glycoprotein VI and Iα2β1 interaction to collagen. Simplagrin binds to RGQOGVMGF peptide with an affinity (KD 11 nM) similar to that of Simplagrin for collagen. Furthermore, Simplagrin prevents laser-induced carotid thrombus formation in vivo without significant bleeding in mice and could be useful as an antithrombotic agent in thrombosis related disease. Conclusion Our results support the orthology of the Aegyptin clade in bloodsucking Nematocera and the hypothesis of a faster evolutionary rate of salivary function of proteins from blood feeding arthropods. PMID:24921659

  11. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet...

  12. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... addition of an aggregating reagent to a platelet-rich plasma. (b) Classification. Class II (performance... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated platelet aggregation system. 864.5700... § 864.5700 Automated platelet aggregation system. (a) Identification. An automated platelet...

  13. Creatine kinase inhibits ADP-induced platelet aggregation

    PubMed Central

    Horjus, D. L.; Nieuwland, R.; Boateng, K. B.; Schaap, M. C. L.; van Montfrans, G. A.; Clark, J. F.; Sturk, A.; Brewster, L. M.

    2014-01-01

    Bleeding risk with antiplatelet therapy is an increasing clinical challenge. However, the inter-individual variation in this risk is poorly understood. We assessed whether the level of plasma creatine kinase, the enzyme that utilizes ADP and phosphocreatine to rapidly regenerate ATP, may modulate bleeding risk through a dose-dependent inhibition of ADP-induced platelet activation. Exogenous creatine kinase (500 to 4000 IU/L, phosphocreatine 5 mM) added to human plasma induced a dose-dependent reduction to complete inhibition of ADP-induced platelet aggregation. Accordingly, endogenous plasma creatine kinase, studied in 9 healthy men (mean age 27.9 y, SE 3.3; creatine kinase 115 to 859 IU/L, median 358), was associated with reduced ADP-induced platelet aggregation (Spearman's rank correlation coefficient, −0.6; p < 0.05). After exercise, at an endogenous creatine kinase level of 4664, ADP-induced platelet aggregation was undetectable, normalizing after rest, with a concomitant reduction of creatine kinase to normal values. Thus, creatine kinase reduces ADP-induced platelet activation. This may promote bleeding, in particular when patients use platelet P2Y12 ADP receptor inhibitors. PMID:25298190

  14. Analysis of aggregation of platelets in thrombosis

    NASA Astrophysics Data System (ADS)

    Ahuja, Suresh

    Platelets are key players in thrombus formation by first rolling over collagen bound von Willebrand factor followed by formation of a stable interaction with collagen. The first adhered platelets bind additional platelets until the whole injury is sealed off by a platelet aggregate. The coagulation system stabilizes the formed platelet plug by creating a tight fibrin network, and then wound contraction takes place because of morphological changes in platelets. Coagulation takes place by platelet activation and aggregation mainly through fibrinogen polymerization into fibrin fibers. The process includes multiple factors, such as thrombin, plasmin, and local shear-rate which regulate and control the process. Coagulation can be divided into two pathways: the intrinsic pathway and the extrinsic pathway. The intrinsic pathway is initiated by the exposure of a negatively charged. It is able to activate factor XII, using a complex reaction that includes prekallikrein and high-molecular-weight kininogen as cofactors.. Thrombin is the final enzyme that is needed to convert fibrinogen into fibrin. The extrinsic pathway starts with the exposure of tissue factor to the circulating blood, which is the major initiator of coagulation. There are several feedback loops that reinforce the coagulation cascade, resulting in large amounts of thrombin. It is dependent on the presence of pro-coagulant surfaces of cells expressing negatively charged phospholipids--which include phosphatidylserine (PS)--on their outer membrane. PS-bearing surfaces are able to increase the efficiency of the reactions by concentrating and co-localizing coagulation factors.. Aggregation of platelets are analyzed and compared to adhesion of platelet to erythrocyte and to endothelial cells. This abstract is replacing MAR16-2015-020003.

  15. Platelet aggregating material from equine arterial tissue

    SciTech Connect

    Schneider, M.D.

    1983-02-22

    Novel hemostatic agent comprises equine arterial fibrillar collagen in a carrier. The agent is useful for the aggregation of platelets for clinical diagnostic tests and for the clotting of blood, such as for controlling bleeding in warm blooded species. The fibrillar collagen is obtained by extracting homogenized equine arterial tissue with aqueous solutions followed by extensive dialysis. No Drawings

  16. Platelet aggregating material from equine arterial tissue

    DOEpatents

    Schneider, Morris D.

    1983-02-22

    Novel hemostatic agent comprises equine arterial fibrillar collagen in a carrier. The agent is useful for the aggregation of platelets for clinical diagnostic tests and for the clotting of blood, such as for controlling bleeding in warm blooded species. The fibrillar collagen is obtained by extracting homogenized equine arterial tissue with aqueous solutions followed by extensive dialysis.

  17. PKC and AKT Modulate cGMP/PKG Signaling Pathway on Platelet Aggregation in Experimental Sepsis

    PubMed Central

    Lopes-Pires, M. Elisa; Naime, Ana C. Antunes; Almeida Cardelli, Nádia J.; Anjos, Débora J.; Antunes, Edson; Marcondes, Sisi

    2015-01-01

    Sepsis severity has been positively correlated with platelet dysfunction, which may be due to elevations in nitric oxide (NO) and cGMP levels. Protein kinase C, Src kinases, PI3K and AKT modulate platelet activity in physiological conditions, but no studies evaluated the role of these enzymes in platelet aggregation in sepsis. In the present study we tested the hypothesis that in sepsis these enzymes positively modulate upstream the NO-cGMP pathway resulting in platelet inhibition. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg, i.p.) and blood was collected after 6 h. Platelet aggregation was induced by ADP (10 μM). Western blotting assays were carried out to analyze c-Src and AKT activation in platelets. Intraplatelet cGMP levels were determined by enzyme immunoassay kit. Phosphorylation of c-SRC at Tyr416 was the same magnitude in platelets of control and LPS group. Incubation of the non-selective Src inhibitor PP2 (10 μM) had no effect on platelet aggregation of LPS-treated rats. LPS increased intraplatelet cGMP levels by 5-fold compared with control group, which was accompanied by 76% of reduction in ADP-induced platelet aggregation. The guanylyl cyclase inhibitor ODQ (25 μM) and the PKG inhibitor Rp-8-Br-PET-cGMPS (25 μM) fully reversed the inhibitory effect of LPS on platelet aggregation. Likewise, the PKC inhibitor GF109203X (10 μM) reversed the inhibition by LPS of platelet aggregation and decreased cGMP levels in platelets. AKT phosphorylation at Thr308 was significantly higher in platelets of LPS compared with control group, which was not reduced by PI3K inhibition. The AKT inhibitor API-1 (20 μM) significantly increased aggregation and reduced cGMP levels in platelets of LPS group. However, the PI3K inhibitor wortmannin and LY29004 had no effect on platelet aggregation of LPS-treated rats. Therefore, inhibition of ADP-induced platelet aggregation after LPS injection is mediated by cGMP/PKG-dependent mechanisms, and PKC and AKT act

  18. Diltiazem potentiates the inhibitory effect of aspirin on platelet aggregation.

    PubMed

    Altman, R; Scazziota, A; Dujovne, C

    1988-09-01

    Platelet aggregation in vivo occurs through the combined effects of many agonists. Aspirin inhibits platelet aggregation but its antiaggregate effects can be overcome by the synergistic action of sodium arachidonate (AA) plus platelet activating factor (PAF). We tested the effect of a calcium entry-blocking agent, diltiazem, on AA-PAF-induced platelet aggregation in platelet-rich plasma from seven healthy volunteers. The studies were done before and after aspirin (100 mg/day) administration for 7 to 10 days. Stimulation of platelet was done in vitro by AA, PAF, or both. Before aspirin treatment, diltiazem (2 micrograms/ml) added in vitro to the platelet-rich plasma inhibited platelet aggregation induced by AA (0.75 mmol/L) by 50%. When PAF was used the inhibition of aggregation was obtained at a lower concentration of diltiazem (0.4 to 1 microgram/ml). After aspirin treatment, AA-induced aggregation was inhibited, and PAF alone (30 nmol/L) produced a first-wave aggregation followed by complete disaggregation. When AA and PAF were added together a full aggregation of postaspirin treatment platelets was obtained. Diltiazem added in vitro at the clinically attainable concentration of 0.1 microgram/ml produced a complete inhibition of this AA-PAF synergism on platelet aggregation. These results suggest that administration of a combination of low-dose aspirin and diltiazem may be of greater benefit than aspirin alone for prophylaxis of cardiovascular diseases where platelets are involved in the pathogenesis.

  19. Platelet aggregation associated with ethanol intoxication

    SciTech Connect

    Volk, S.; Walenga, J.; Fareed, J.; Schumacher, H. )

    1989-02-09

    Alcohol is known to produce profound effects on blood; during chronic intoxication, prolongation of bleeding time has been reported. Utilizing human platelet rich plasma, we have studied the effect of alcohol on epinephrine, arachidonic acid and ADP induced aggregation. Control responses were obtained with saline from which the relative inhibition by alcohol was calculated. These studies were carried out at a concentration of 1.25-5.0 mg/ml which represents 0.125-0.5% alcohol blood levels. From 25 normal male and female volunteers, without prior hemostatic defects or drug ingestion, a dose-dependent inhibition by alcohol of all three agonist induced aggregations was noted. Alcohol itself did not produce any aggregation response. These studies demonstrate that alcohol at levels which are reached during intoxication is capable of impairing platelet function. The implication of this finding on the bleeding complications in healthy intoxicated patients may be significant during traumatic events, and individuals taking antiplatelet drugs may present a more serious hemostatic deficit during alcohol intoxication.

  20. The effect of ex vivo anticoagulants on whole blood platelet aggregation.

    PubMed

    Kalb, Madeleine L; Potura, Lukasz; Scharbert, Gisela; Kozek-Langenecker, Sibylle A

    2009-02-01

    Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended.

  1. In vitro canine platelet aggregation caused by Dirofilaria immitis extract

    PubMed Central

    TAKASHIMA, Yasuhiro; ONODA, Isako; CHIOU, Shin-Pin; KITOH, Katsuya

    2016-01-01

    Platelet function hyper-activity has been reported in Dirofilaria immitis (heartworm, HW)-infected dogs. Although the mechanism of increased platelet hyper-activity has not yet been elucidated, it is suggested to be mediated by unknown factors, which may be related to adult HW components. This study aims to determine whether adult male HW whole body extract induces canine platelet aggregation in vitro. The results indicate that HW extract caused an aggregation of canine platelets in a concentration-dependent manner. This aggregation ability of the HW extract was not mediated by the adenosine diphosphate receptor. In addition, the mechanisms of aggregation did not require cyclooxygenase-dependent pathways, and the aggregating activity of substances contained in the HW extract was heat stable; therefore, the active substances may be different from collagen. Furthermore, the platelet aggregating activity remained within the molecular weight (MW)≥100,000 fraction obtained by ultrafiltrating the HW extract. In contrast, the MW <100,000 fraction also had a platelet aggregation ability, but the aggregation pattern was reversible and the maximum extent decreased, compared with the MW≥100,000 fraction response. Our experiments have been conducted using a whole body extract from adult HWs to determine with certainty the aggregating activity of HW elements on canine platelets. More studies are necessary to evaluate the effects of the metabolic products released from live adult worms in pulmonary arteries and the symbiont bacterium Wolbachia-derived antigens on canine platelet aggregation. PMID:28049921

  2. In vitro canine platelet aggregation caused by Dirofilaria immitis extract.

    PubMed

    Takashima, Yasuhiro; Onoda, Isako; Chiou, Shin-Pin; Kitoh, Katsuya

    2017-02-28

    Platelet function hyper-activity has been reported in Dirofilaria immitis (heartworm, HW)-infected dogs. Although the mechanism of increased platelet hyper-activity has not yet been elucidated, it is suggested to be mediated by unknown factors, which may be related to adult HW components. This study aims to determine whether adult male HW whole body extract induces canine platelet aggregation in vitro. The results indicate that HW extract caused an aggregation of canine platelets in a concentration-dependent manner. This aggregation ability of the HW extract was not mediated by the adenosine diphosphate receptor. In addition, the mechanisms of aggregation did not require cyclooxygenase-dependent pathways, and the aggregating activity of substances contained in the HW extract was heat stable; therefore, the active substances may be different from collagen. Furthermore, the platelet aggregating activity remained within the molecular weight (MW)≥100,000 fraction obtained by ultrafiltrating the HW extract. In contrast, the MW <100,000 fraction also had a platelet aggregation ability, but the aggregation pattern was reversible and the maximum extent decreased, compared with the MW≥100,000 fraction response. Our experiments have been conducted using a whole body extract from adult HWs to determine with certainty the aggregating activity of HW elements on canine platelets. More studies are necessary to evaluate the effects of the metabolic products released from live adult worms in pulmonary arteries and the symbiont bacterium Wolbachia-derived antigens on canine platelet aggregation.

  3. [Mechanism of cooked blanched garlic leaves against platelet aggregation].

    PubMed

    Wang, Xin-Hua; Di, Yan-Hui

    2014-06-01

    This study was purposed to explore the mechanism of cooked blanched garlic leave juice against platelet aggregation. The juice of blanched garlic leaves was mixed with platelet rich plasma (PRP), the human platelet aggregation, the activation of human platelets induced by adenosine diphosphate (ADP) and collagen were observed; the expression levels of the activated platelets (Fib-R) and P-selectin (CD62P), and the amount of platelet fibrinogen binding were detected by flow cytometry; 10 rabbits were randomly divided into two groups, in addition to the normal diet, they were fed with physiologic saline and cooked blanched garlic leave juice respectively. After 1, 3, 5 , 8 weeks, the maximum ratio of rabbit platelet aggregation induced by ADP and collagen were observed . The results showed that the cooked blanched garlic leave juice could significantly inhibit human platelet aggregation induced by ADP and collagen (P < 0.05), the inhibitory ratio were 87.37% and 86.24% respectively; the juice could not inhibit activated platelets Fib-R and CD62P expression levels (P > 0.05), but was able to inhibit platelet fibrinogen binding capacity (P < 0.05); the rabbit platelet aggregation rate in the group given cooked blanched garlic leave juice was significantly lower than that in control group (P < 0.05). It is concluded that the cooked blanched garlic leave juice can inhibit platelet aggregation in vitro and in vivo, the inhibition of aggregation pathway mainly is blocking the combination of fibrinogen with Fib-R, which finally results in the inhibition of platelet aggregation. Therefore, regular consumption of cooked blanched garlic leaves may prevent cardiovascular thrombotic diseases.

  4. 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.

  5. Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow

    PubMed Central

    De Angelis, Valentina; Koekman, Arnold C.; Weeterings, Cees; Roest, Mark; de Groot, Philip G.; Herczenik, Eszter; Maas, Coen

    2014-01-01

    Background The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells. Additionally, it has been reported that endocannabinoid receptors are present on circulating platelets, but there has been conflicting evidence on their contribution to platelet function. Objectives Our aim was to examine the role of endocannabinoids in platelet function in vitro and in vivo. Methods and Results We studied the effects of the well-characterized endogenous endocannabinoid anandamide on platelet aggregation in suspension, α-granule release, calcium mobilization, Syk phosphorylation, as well as platelet spreading and aggregate formation under flow. Anandamide inhibits platelet aggregation and α-granule release by collagen, collagen-derived peptide CRP-XL, ADP, arachidonic acid and thromboxane A2 analogue U46619. However, activation via thrombin receptor PAR-1 stays largely unaffected. Calcium mobilization is significantly impaired when platelets are stimulated with collagen or CRP-XL, but remains normal in the presence of the other agonists. In line with this finding, we found that anandamide prevents collagen-induced Syk phosphorylation. Furthermore, anandamide-treated platelets exhibit reduced spreading on immobilized fibrinogen, have a decreased capacity for binding fibrinogen in solution and show perturbed platelet aggregate formation under flow over collagen. Finally, we investigated the influence of Cannabis sativa consumption by human volunteers on platelet activation. Similar to our in vitro findings with anandamide, ex vivo collagen-induced platelet aggregation and aggregate formation on immobilized collagen under flow were impaired in whole blood of donors that had consumed Cannabis sativa. Conclusions Endocannabinoid receptor agonists reduce platelet activation and aggregate formation both in vitro and ex vivo after Cannabis sativa consumption. Further elucidation of this novel regulatory mechanism for platelet function

  6. Constitutively activated phosphatidylinositol 3-kinase primes platelets from patients with chronic myelogenous leukemia for thrombopoietin-induced aggregation.

    PubMed

    Kubota, Y; Tanaka, T; Ohnishi, H; Kitanaka, A; Okutani, Y; Taminato, T; Ishida, T; Kamano, H

    2004-06-01

    In this study, we examined the effect of thrombopoietin (TPO) on the aggregation of platelets from 40 patients with myeloproliferative disorders (MPDs), including 17 patients with chronic myelogenous leukemia in the chronic phase (CML-CP), 10 with polycythemia vera, 10 with essential thrombocythemia, and three with myelofibrosis. TPO by itself dose-dependently induced the aggregation of platelets from patients with CML-CP but not from those with other MPDs or with CML-CP in cytogenetical complete remission. The expression of CD63 in CML-CP platelets was induced by TPO treatment. Phosphatidylinositol 3-kinase (PI3-kinase) was constitutively activated in CML-CP platelets. Pretreatment with PI3-kinase inhibitors (wortmannin and LY294002) dose-dependently inhibited TPO-induced aggregation of CML-CP platelets. The Abl kinase inhibitor imatinib mesylate and the Jak inhibitor AG490 suppressed TPO-induced aggregation of CML-CP platelets. Pretreatment with imatinib mesylate, but not with AG490, inhibited the activity of PI3-kinase in CML-CP platelets. In addition, tyrosine phosphorylation of Jak2 was undetected in CML-CP platelets before TPO treatment. These findings indicate that the constitutive activation of PI3-kinase primes CML-CP platelets for the aggregation induced by TPO, and that Bcr-Abl, but not Jak family protein tyrosine kinases, are involved in the constitutive activation of PI3-kinase in CML-CP platelets.

  7. COX, LOX and platelet aggregation inhibitory properties of Lauraceae neolignans.

    PubMed

    Coy, Ericsson David; Cuca, Luis Enrique; Sefkow, Michael

    2009-12-15

    The anti-inflammatory potential of 26 neolignans (14 of the bicyclooctane-type and 12 of the benzofuran-type), isolated from three Lauraceae species (Pleurothyrium cinereum, Ocotea macrophylla and Nectandra amazonum), was evaluated in vitro through inhibition of COX-1, COX-2, 5-LOX and agonist-induced aggregation of rabbit platelets. Benzofuran neolignans were found to be selective COX-2 inhibitors, whereas bicyclooctane neolignans inhibit selectively the PAF-action as well as COX-1 and 5-LOX. The neolignan 9-nor-7,8-dehydro-isolicarin B 15 and cinerin C 7 were found to be the most potent COX-2 inhibitor and PAF-antagonist, respectively. Nectamazin C 10 exhibited dual 5-LOX/COX-2 inhibition.

  8. Crystallization and preliminary X-ray diffraction studies of BmooPLA2-I, a platelet-aggregation inhibitor and hypotensive phospholipase A2 from Bothrops moojeni venom

    PubMed Central

    Salvador, Guilherme H. M.; Marchi-Salvador, Daniela P.; Silveira, Lucas B.; Soares, Andreimar M.; Fontes, Marcos R. M.

    2011-01-01

    Phospholipases A2 (PLA2s) are enzymes that cause the liberation of fatty acids and lysophospholipids by the hydrolysis of membrane phospholipids. In addition to their catalytic action, a wide variety of pharmacological activities have been described for snake-venom PLA2s. BmooPLA2-I is an acidic, nontoxic and catalytic PLA2 isolated from Bothrops moojeni snake venom which exhibits an inhibitory effect on platelet aggregation, an immediate decrease in blood pressure, inducing oedema at a low concentration, and an effective bactericidal effect. BmooPLA2-I has been crystallized and X-ray diffraction data have been collected to 1.6 Å resolution using a synchrotron-radiation source. The crystals belonged to space group C2221, with unit-cell parameters a = 39.7, b = 53.2, c = 89.2 Å. The molecular-replacement solution of BmooPLA2-I indicated a monomeric conformation, which is in agreement with nondenaturing electrophoresis and dynamic light-scattering experiments. A comparative study of this enzyme with the acidic PLA2 from B. jararacussu (BthA-I) and other toxic and nontoxic PLA2s may provide important insights into the functional aspects of this class of proteins. PMID:21821890

  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

    Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab

  10. Effects of Suilysin on Streptococcus suis-Induced Platelet Aggregation

    PubMed Central

    Zhang, Shengwei; Wang, Junping; Chen, Shaolong; Yin, Jiye; Pan, Zhiyuan; Liu, Keke; Li, Lin; Zheng, Yuling; Yuan, Yuan; Jiang, Yongqiang

    2016-01-01

    Blood platelets play important roles during pathological thrombocytopenia in streptococcal toxic shock syndrome (STSS). Streptococcus suis (S. suis) an emerging human pathogen, can cause STSS similarly to S. pyogenes. However, S. suis interactions with platelets are poorly understood. Here, we found that suilysin (SLY), different from other bacterial cholesterol-dependent cytolysins (CDCs), was the sole stimulus that induced platelet aggregation. Furthermore, the inside-out activation of GPIIb/IIIa of platelets mediated SLY-induced platelet aggregation. This process was triggered by Ca2+ influx that depend on the pore forming on platelets by SLY. Additionally, although SLY induced α-granule release occurred via the MLCK-dependent pathway, PLC-β-IP3/DAG-MLCK and Rho-ROCK-MLCK signaling were not involved in SLY-induced platelet aggregation. Interestingly, the pore dependent Ca2+ influx was also found to participate in the induction of platelet aggregation with pneumolysin (PLY) and streptolysin O (SLO), two other CDCs. It is possible that the CDC-mediated platelet aggregation we observed in S. suis is a similar response mechanism to that used by a wide range of bacteria. These findings might lead to the discovery of potential therapeutic targets for S. suis-associated STSS. PMID:27800304

  11. CGX1037 is a novel PKC isoform delta selective inhibitor in platelets.

    PubMed

    Bhavanasi, Dheeraj; Kostyak, John C; Swindle, John; Kilpatrick, Laurie E; Kunapuli, Satya P

    2015-01-01

    Platelets upon activation change their shape, aggregate and secrete alpha and dense granule contents among which ADP acts as a feedback activator. Different Protein Kinase C (PKC) isoforms have specific non-redundant roles in mediating platelet responses including secretion and thrombus formation. Murine platelets lacking specific PKC isoforms have been used to evaluate the isoform specific functions. Novel PKC isoform δ has been shown to play an important role in some pathological processes. Lack of specific inhibitors for PKCδ has restricted analysis of its role in various cells. The current study was carried out to evaluate a novel small molecule PKCδ inhibitor, CGX1037 in platelets. Platelet aggregation, dense granule secretion and western blotting experiments were performed to evaluate CGX1037. In human platelets, CGX1037 inhibited PAR4-mediated phosphorylation on PKD2, a PKCδ-specific substrate. Pre-treatment of human or murine platelets with CGX1037 inhibited PAR4-mediated dense granule secretion whereas it potentiated GPVI-mediated dense granule secretion similar to the responses observed in murine platelets lacking PKCδ· Furthermore, pre-treatment of platelets from PKCδ(-/-) mice with CGX1037 had no significant additive effect on platelet responses suggesting the specificity of CGX1037. Hence, we show that CGX1037 is a selective small molecule inhibitor of PKCδ in platelets.

  12. Effects of alcohol ingestion post-exercise on platelet aggregation.

    PubMed

    El-Sayed, Mahmoud S

    2002-01-15

    The present study examined the influence of ingesting a moderate dose of alcohol on platelet count and platelet aggregation during recovery following exercise. Nineteen subjects (11 male and 8 female) were studied immediately after a standardised cycle ergometer test and during the 24-h period of recovery. In random order, alcohol (0.7 g/kg body mass) was given 1 h after exercise on one test occasion, while an equal volume of alcohol-free solution was administered on the other. Venous blood samples were obtained at baseline, post-exercise, and at 1, 5, and 22 h post-alcohol ingestion. Blood alcohol level increased significantly 1 h after the ingestion of alcohol, but decreased and returned to the resting baseline level at 5 h during recovery. Males and females subjects exhibited similar mean values of platelet count, platelet aggregation, and beta-thromboglobulin concentration at rest and following exercise and recovery. A significant increase in platelet count and a decrease in platelet aggregation using adenosine diphosphate (ADP) was found following exercise. Although plasma beta-thromboglobulin level (pooled data for males and females) showed an increase by 26.0% (from a mean pre-exercise value of 22.3-28.1 IU/ml), this rise was not significant (P>.05). The post-exercise increase in platelet count was mainly due to exercise-induced plasma volume loss. During recovery, while the increase in platelet count post-exercise returned to the baseline level in control and alcohol trials, the optical density of platelet aggregation remained significantly depressed at 5-h during recovery in the alcohol trial but not in the normal control condition. It is concluded that exercise induces significant reduction in platelet aggregation and the consumption of alcohol after physical exercise delays the normal return of platelet aggregation to the resting baseline levels during recovery.

  13. Platelet aggregability in relation to impaired consciousness after head injury.

    PubMed Central

    Vecht, C J; Minderhoud, J M; Sibinga, C T

    1975-01-01

    ADP-induced platelet aggregation was studied for up to six weeks in 34 patients with head injuries. The patients were divided into three groups according to the degree of impaired consciousness assessed by a clinical coma scale, and change in platelet aggregation was related to the coma score. Platelet aggregation was markedly reduced in all eight patients dying within 24 hours of injury. All 17 patients who remained unconscious for four days or more showed decreased platelet aggregation up to nine days after admission, the most marked effect being on the second day. Platelet function in this group returned to normal within 16 days. Nine patients with only slightly impaired consciousness also showed subnormal platelet aggregation during the first few days with a return to normal by the fourth day. Platelet counts remained within normal limits in all groups. We suggest that during coma following head injury brainstem dysfunction induces neurohumoral changes in the blood which are responsible for a decrease in platelet function. PMID:1214015

  14. Synthetic polyphosphate inhibits endogenous coagulation and platelet aggregation in vitro

    PubMed Central

    Yang, Xiaoyang; Wan, Mengjie; Liang, Ting; Peng, Minyuan; Chen, Fangping

    2017-01-01

    Platelet-derived polyphosphate has previously been indicated to induce coagulation. However, industrially synthesized polyphosphate has been found to have different effects from those of the platelet-derived form. The present study investigated whether synthetic sodium polyphosphate inhibits coagulation using routine coagulation tests and thromboelastography. Synthetic polyphosphate was found to inhibit adenosine diphosphate-, epinephrine-, arachidonic acid-, ristocetin-, thrombin-, oxytocin- and pituitrin-induced platelet aggregation. The effects of synthetic polyphosphate in clotting inhibition were revealed by the analysis of clotting factor activity and platelet aggregation tests. Synthetic polyphosphate may inhibit platelet aggregation by reducing platelet calcium levels, as indicated by the results of flow cytometric analysis and high-throughput fluorescent screening. Furthermore, analysis of thromboxane (TX)B2 by ELISA indicated that synthetic polyphosphate reduces platelet aggregation by inhibiting the TXA2 signaling pathway. In conclusion, synthetic polyphosphate inhibits clotting factor activity and endogenous coagulation by reducing the levels of calcium ions and TXA2 to curb platelet aggregation. PMID:28123708

  15. An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets.

    PubMed

    Sánchez Guiu, I M; Martínez-Martinez, I; Martínez, C; Navarro-Fernandez, J; García-Candel, F; Ferrer-Marín, F; Vicente, V; Watson, S P; Andrews, R K; Gardiner, E E; Lozano, M L; Rivera, J

    2015-08-01

    Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donor citrated platelet-rich plasma (PRP), but not in PRP from Glanzmann's thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, (14)C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

  16. Angiotensin II AT1 receptor antagonists inhibit platelet adhesion and aggregation by nitric oxide release.

    PubMed

    Kalinowski, Leszek; Matys, Tomasz; Chabielska, Ewa; Buczko, Włodzimierz; Malinski, Tadeusz

    2002-10-01

    This study investigated the process of nitric oxide (NO) release from platelets after stimulation with different angiotensin II type 1 (AT1)-receptor antagonists and its effect on platelet adhesion and aggregation. Angiotensin II AT1-receptor antagonist-stimulated NO release in platelets was compared with that in human umbilical vein endothelial cells by using a highly sensitive porphyrinic microsensor. In vitro and ex vivo effects of angiotensin II AT1-receptor antagonists on platelet adhesion to collagen and thromboxane A2 analog U46619-induced aggregation were evaluated. Losartan, EXP3174, and valsartan alone caused NO release from platelets and endothelial cells in a dose-dependent manner in the range of 0.01 to 100 micro mol/L, which was attenuated by NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. The angiotensin II AT1-receptor antagonists had more than 70% greater potency in NO release in platelets than in endothelial cells. The degree of inhibition of platelet adhesion (collagen-stimulated) and aggregation (U46619-stimulated) elicited by losartan, EXP3174, and valsartan, either in vitro or ex vivo, closely correlated with the NO levels produced by each of these drugs alone. The inhibiting effects of angiotensin II AT1-receptor antagonists on collagen-stimulated adhesion and U46619-stimulated aggregation of platelets were significantly reduced by pretreatment with N(G)-nitro-L-arginine methyl ester. Neither the AT2 receptor antagonist PD123319, the cyclooxygenase synthase inhibitor indomethacin, nor the selective thromboxane A2/prostaglandin H2 receptor antagonist SQ29,548 had any effect on angiotensin II AT1-receptor antagonist-stimulated NO release in platelets and endothelial cells. The presented studies clearly indicate a crucial role of NO in the arterial antithrombotic effects of angiotensin II AT1-receptor antagonists.

  17. Acid soluble platelet aggregating material isolated from human umbilical cord

    SciTech Connect

    Schneider, M.D.

    1983-12-27

    An acid soluble, pepsin sensitive platelet aggregating material is isolated from human umbilical cord tissue by extraction with dilute aqueous acid. The method of isolation is disclosed and its use to control bleeding is described. 2 figs.

  18. [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.

  19. Lysophosphatidic acids. Influence on platelet aggregation and intracellular calcium flux.

    PubMed Central

    Gerrard, J. M.; Kindom, S. E.; Peterson, D. A.; Peller, J.; Krantz, K. E.; White, J. G.

    1979-01-01

    Decanoyl-, palmitoyl-, and oleoyl-lysophosphatidic acid (LPA) were studied for their effects on platelet aggregation and intracellular calcium flux. Palmitoyl-LPA and oleoyl-LPA both caused a concentration-dependent aggregation of human blood platelets at concentrations of 12--300 microM. Aggregation by adenosine diphosphate (ADP) was enhanced at slightly lower concentrations. First-wave aggregation induced by these LPAs was not blocked by aspirin, indomethacin, or heparin, suggesting similarities to ADP aggregation. However, in washed platelets with a high calcium concentration, no serotonin secretion was observed, even though full aggregation occurred, suggesting that aggregation was not due to released ADP. This concept was supported by studies of platelets deficient in the storage pool of ADP and serotonin, which had a normal first-wave aggregation response to palmitoyl-LPA. Aggregation induced by palmitoyl LPA was inhibited by prostaglandin E1 (PGE1), theophylline, and ethylenediaminotetraacetate (EDTA), though in the presence of EDTA shape change occurred. Aggregation stimulated by palmitoyl-LPA or oleoyl-LPA was characterized by changes in the shape of the platelets with development of pseudopods and centralization of granules closely surrounded by contractile microfilaments and supporting microtubules. The addition of palmitoyl-LPA and oleoyl-LPA, but not decanoyl-LPA, caused the release of calcium from a platelet membrane fraction that contains elements of the intracellular calcium storage system and actively concentrates this cation in the presence of adenosine triphosphate (ATP) and magnesium. It is suggested that LPAs cause aggregation by stimulating the release of calcium intracellularly. Images Figure 1 Figure 2 Figure 3 Figure 4 Text-Figure 6 PMID:112871

  20. A simple method for activating the platelets used in microfluidic platelet aggregation tests: Stirring-induced platelet activation

    PubMed Central

    Lee, Hoyoon; Kim, Gyehyu; Lim, Chaeseung; Lee, ByoungKwon; Shin, Sehyun

    2016-01-01

    High-shear stimulation is well known as one of the key factors affecting platelet activation and aggregation, which can lead to the formation of a thrombus. In one of our previous studies, we introduced migration distance-based platelet function analysis in a microfluidic system. In this study, we set out to examine the effects of stirring on shear-induced platelet activation and aggregation in a chamber system by using a rotating stirrer. We found that the rotating stirrer caused not only rotational shear flow but also a strong radial secondary flow. The latter flow led to efficient mixing in the chamber. Moreover, the rotational flow led to the generation of shear stress, the magnitude of which can be controlled to activate the platelets. Activated platelets tend to aggregate themselves. The maximum platelet aggregation was observed at a critical shear rate of 3100 s−1, regardless of the stirrer shape. Furthermore, the time taken to attain maximum aggregation was significantly shortened when using a wide stirrer (30 s) instead of a narrow one (180 s). When using a flat stirrer, the non-uniform shear field in the chamber system was resolved with the radial secondary flow-induced mixing; thus, most of the platelets were homogenously activated. The stirring-induced platelet activation mechanism was experimentally confirmed in a microfluidic system for a platelet aggregation test while monitoring the migration distance until the microfluidic channel is occluded. Our findings indicate that the present system, consisting of a rotating stirrer and a confined chamber, provides effective shear stimulation for activating platelets and inducing platelet aggregates. PMID:28058084

  1. A simple method for activating the platelets used in microfluidic platelet aggregation tests: Stirring-induced platelet activation.

    PubMed

    Lee, Hoyoon; Kim, Gyehyu; Lim, Chaeseung; Lee, ByoungKwon; Shin, Sehyun

    2016-11-01

    High-shear stimulation is well known as one of the key factors affecting platelet activation and aggregation, which can lead to the formation of a thrombus. In one of our previous studies, we introduced migration distance-based platelet function analysis in a microfluidic system. In this study, we set out to examine the effects of stirring on shear-induced platelet activation and aggregation in a chamber system by using a rotating stirrer. We found that the rotating stirrer caused not only rotational shear flow but also a strong radial secondary flow. The latter flow led to efficient mixing in the chamber. Moreover, the rotational flow led to the generation of shear stress, the magnitude of which can be controlled to activate the platelets. Activated platelets tend to aggregate themselves. The maximum platelet aggregation was observed at a critical shear rate of 3100 s(-1), regardless of the stirrer shape. Furthermore, the time taken to attain maximum aggregation was significantly shortened when using a wide stirrer (30 s) instead of a narrow one (180 s). When using a flat stirrer, the non-uniform shear field in the chamber system was resolved with the radial secondary flow-induced mixing; thus, most of the platelets were homogenously activated. The stirring-induced platelet activation mechanism was experimentally confirmed in a microfluidic system for a platelet aggregation test while monitoring the migration distance until the microfluidic channel is occluded. Our findings indicate that the present system, consisting of a rotating stirrer and a confined chamber, provides effective shear stimulation for activating platelets and inducing platelet aggregates.

  2. Further insights into the anti-aggregating activity of NMDA in human platelets

    PubMed Central

    Franconi, Flavia; Miceli, Mauro; Alberti, Luisa; Seghieri, Giuseppe; De Montis, M Graziella; Tagliamonte, Alessandro

    1998-01-01

    In the present study the effect of N-methyl-D-aspartate (NMDA) on thromboxane B2 synthesis and on [Ca2+]i was studied in human platelets.NMDA (10−7 M) completely inhibited the synthesis of thromboxane B2 from exogenous arachidonic acid (AA), while it did not interfere with the aggregating effect of the thromboxane A2 receptor agonist U-46619.NMDA (0.1 μM–10 μM) dose-dependently increased intracellular calcium in washed platelets pre-loaded with fura 2 AM, and this effect was not additive with that of AA.NMDA shifted the dose-response curve of AA to the right. At the highest AA concentrations platelet aggregation was not inhibited.The antiaggregating effect of NMDA was not antagonized by NG-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor.Finally, NMDA (0.01 nM–100 nM) associated with either aspirin or indomethacin significantly potentiated the antiaggregating activity of both cyclo-oxygenase inhibitors.It was concluded that NMDA is a potent inhibitor of platelet aggregation and thromboxane B2 synthesis in human platelet rich plasma (PRP). PMID:9630340

  3. Keishibukuryogan, a Traditional Japanese Medicine, Inhibits Platelet Aggregation in Guinea Pig Whole Blood

    PubMed Central

    Terawaki, Kiyoshi; Noguchi, Masamichi; Yuzurihara, Mitsutoshi; Omiya, Yuji; Ikarashi, Yasushi; Kase, Yoshio

    2015-01-01

    Effects of keishibukuryogan (KBG) on platelet aggregation were investigated. To ensure the specificity of KBG, tokishakuyakusan (TSS) and kamisyoyosan (KSS), which are known to have platelet aggregation-inhibiting effects, and rikkunshito (RKT) and shakuyakukanzoto (SKT), which are considered to be devoid of such effects, were used for comparison. The platelet aggregation of each test drug was measured by the screen filtration pressure method using whole blood of guinea pigs and expressed as a collagen-induced pressure rate (%) or a collagen concentration required for 50% increase in the pressure rate (PATI value). KBG suppressed the collagen-induced whole blood pressure rate increase and increased the PATI value, like TSS and KSS. Neither RKT nor SKT showed these effects. The Moutan cortex and Cinnamomi cortex, the constituent crude drugs of KBG, showed KBG-like pressure rate suppression and PATI-increasing effects. Furthermore, paeonol, a representative component of Moutan cortex, and aspirin which is known to have platelet aggregation-inhibiting activity (COX-1 inhibitor) also showed similar effects. These results suggest that the platelet aggregation-inhibiting activity of the constituent crude drugs Moutan cortex and Cinnamomi cortex is involved in the improving effects of KBG on impaired microcirculation and that paeonol plays a role in these effects. PMID:26379740

  4. Inhibition of rat platelet aggregation by Urtica dioica leaves extracts.

    PubMed

    El Haouari, Mohammed; Bnouham, Mohamed; Bendahou, Mourad; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Mekhfi, Hassane

    2006-07-01

    Platelet hyperactivity plays an important role in arterial thrombosis and atherosclerosis. The present study was undertaken to investigate the effects of different extracts of Urtica dioica leaves on platelet aggregation. Rat platelets were prepared and incubated in vitro with different concentrations of the tested extracts and aggregation was induced by different agonists including thrombin (0.5 U/mL), ADP (10 microm), epinephrine (100 microm) and collagen (5 mg/mL). The crude aqueous extract inhibited thrombin-induced platelet aggregation in a dose-dependent manner. At 1 mg/mL, the percent inhibition was 17.1 +/- 4.2%. Soxhlet extraction of the plant leaves with different successive solvents showed that the ethyl acetate extract exhibited the most antiaggregant effect with an inhibition of 76.8 +/- 6.1% at 1 mg/mL. Flavonoids isolated from the plant leaves, produced a strong inhibitory effect on thrombin-induced platelet aggregation with an IC(50) of 0.25 +/- 0.05 and 0.40 +/- 0.04 mg/mL for genins and heterosidic flavonoids, respectively. Flavonoids also markedly inhibited platelet aggregation induced by ADP, collagen and epinephrine. It is concluded that Urtica dioica has an antiplatelet action in which flavonoids are mainly implicated. These results support the traditional use of Urtica dioica in the treatment and/or prevention of cardiovascular disease.

  5. Effects of nitroblue tetrazolium and vitamin E on platelet ultrastructure, aggregation, and secretion

    PubMed Central

    White, James G.; Rao, Gundu H. R.; Gerrard, Jonathan M.

    1977-01-01

    All agents capable of triggering the platelet release reaction also stimulate prostaglandin biosynthesis in these cells. Information concerning the endoperoxides, thromboxanes, and more stable metabolites generated by the action of cyclooxygenase and lipoxygenase on arachidonic acid has accumulated rapidly, but little is known about the preliminary steps in the cleavage and preparation of arachidonic acid for insertion into the enzymatic pathways of prostaglandin synthesis. Studies in this laboratory have shown that the combination of nitroblue tetrazolium (NBT) and vitamin E which prevents oxygenation of arachidonic acid to a free radical also blocks platelet prostaglandin biosynthesis. The present study has evaluated the influence of NBT, vitamin E, and the combination of NBT and vitamin E on the fine structure and biochemistry of platelets during incubation, and the effects of these compounds on the aggregation and secretion of platelets stimulated by collagen, thrombin, epinephrine, and ADP. Results of the study demonstrate that NBT and vitamin E, rather than injuring platelets, appear to protect them during incubation. Together NBT and vitamin E blocked aggregation by epinephrine, collagen, and thrombin, but permitted a small first wave stimulated by ADP. Both ADP and thrombin induced shape change, pseudopod formation, and limited degrees of internal contraction in vitamin E-NBT-treated platelets, whereas epinephrine and collagen failed to significantly alter discoid form. This pattern of response to aggregating agents was identical to reactions observed in platelets pretreated with aspirin and indomethacin, both potent inhibitors of platelet prostaglandin synthesis. In addition, NBT-vitamin E virtually blocked the first wave of aggregation which is not affected by aspirin and indomethacin. The findings support the concept that conversion of arachidonic acid to an activated state is an important step in prostaglandin synthesis and that electron transfer or

  6. Platelet-cytokine Complex Suppresses Tumour Growth by Exploiting Intratumoural Thrombin-dependent Platelet Aggregation

    PubMed Central

    Li, Yu-Tung; Nishikawa, Tomoyuki; Kaneda, Yasufumi

    2016-01-01

    Tumours constitute unique microenvironments where various blood cells and factors are exposed as a result of leaky vasculature. In the present study, we report that thrombin enrichment in B16F10 melanoma led to platelet aggregation, and this property was exploited to administer an anticancer cytokine, interferon-gamma induced protein 10 (IP10), through the formation of a platelet-IP10 complex. When intravenously infused, the complex reached platelet microaggregates in the tumour. The responses induced by the complex were solely immune-mediated, and tumour cytotoxicity was not observed. The complex suppressed the growth of mouse melanoma in vivo, while both platelets and the complex suppressed the accumulation of FoxP3+ regulatory T cells in the tumour. These results demonstrated that thrombin-dependent platelet aggregation in B16F10 tumours defines platelets as a vector to deliver anticancer cytokines and provide specific treatment benefits. PMID:27117228

  7. Enhancement of Platelet Aggregation by Ursolic Acid and Oleanolic Acid

    PubMed Central

    Kim, Mikyung; Han, Chang-ho; Lee, Moo-Yeol

    2014-01-01

    The pentacyclic triterpenoid ursolic acid (UA) and its isomer oleanolic acid (OA) are ubiquitous in food and plant medicine, and thus are easily exposed to the population through natural contact or intentional use. Although they have diverse health benefits, reported cardiovascular protective activity is contentious. In this study, the effect of UA and OA on platelet aggregation was examined on the basis that alteration of platelet activity is a potential process contributing to cardiovascular events. Treatment of UA enhanced platelet aggregation induced by thrombin or ADP, which was concentration-dependent in a range of 5–50 μM. Quite comparable results were obtained with OA, in which OA-treated platelets also exhibited an exaggerated response to either thrombin or ADP. UA treatment potentiated aggregation of whole blood, while OA failed to increase aggregation by thrombin. UA and OA did not affect plasma coagulation assessed by measuring prothrombin time and activated partial thromboplastin time. These results indicate that both UA and OA are capable of making platelets susceptible to aggregatory stimuli, and platelets rather than clotting factors are the primary target of them in proaggregatory activity. These compounds need to be used with caution, especially in the population with a predisposition to cardiovascular events. PMID:25009707

  8. Platelets and atherogenesis: Platelet anti-aggregation activity and endothelial protection from tomatoes (Solanum lycopersicum L.)

    PubMed Central

    PALOMO, IVÁN; FUENTES, EDUARDO; PADRÓ, TERESA; BADIMON, LINA

    2012-01-01

    In recent years, it has been shown that platelets are not only involved in the arterial thrombotic process, but also that they play an active role in the inflammatory process of atherogenesis from the beginning. The interaction between platelets and endothelial cells occurs in two manners: activated platelets unite with intact endothelial cells, or platelets in resting adhere to activated endothelium. In this context, inhibition of the platelet function (adhesion/aggregation) could contribute to the prevention of atherothrombosis, the leading cause of cardiovascular morbidity. This can be achieved with antiplatelet agents. However, at the public health level, the level of primary prevention, a healthy diet has also been shown to exert beneficial effects. Among those elements of a healthy diet, the consumption of tomatoes (Solanum lycopersicum L.) stands out for its effect on platelet anti-aggregation activity and endothelial protection, which may be beneficial for cardiovascular health. This article briefly discusses the involvement of platelets in atherogenesis and the possible mechanisms of action provided by tomatoes for platelet anti-aggregation activity and endothelial protection. PMID:22969932

  9. Molecular determinants of the platelet aggregation inhibitory activity of carbamoylpiperidines.

    PubMed

    Feng, Z; Gollamudi, R; Dillingham, E O; Bond, S E; Lyman, B A; Purcell, W P; Hill, R J; Korfmacher, W A

    1992-08-07

    A series of alpha,alpha'-bis[3-(N,N-dialkylcarbamoyl)piperidino]-p- xylenes were synthesized and tested for their inhibitory activity on ADP-induced aggregation of human platelets. A parabolic curve was obtained when log 1/C (activity) was plotted against log P (octanol/water partition coefficient). Using this as a model, a new analogue, alpha,alpha'-bis-[3-(N-methyl-N-butylcarbamoyl)piperidino]-p-xylen e (3g), was synthesized with a predicted IC50 of 25 microM. When this compound was subsequently evaluated, the IC50 was 22.1 +/- 5.5 microM, demonstrating the applicability of this model. The amide oxygen of the carbamoyl substituent appeared necessary for activity. Thus, for example, when the amide carbonyl group of 3a (IC50 = 44.5 microM) was reduced to CH2, the resulting compound 4 had a dramatically reduced activity, IC50 = 1565 microM. Compound 3a was resolved into (+) and (-) enantiomers and a meso (0) diastereomer using fractional crystallization, diastereomeric tartrate formation, and chiral HPLC. Compared to (-)-3a, the (+) isomer was 15 times more potent when ADP was the agonist and 19 times more active when collagen was used as the agonist. Molecular modeling of R,R- and S,S-3a using the SYBYL program was used to examine their interactions with phosphatidylinositol (PI). There was a better fit between PI and the R,R-3a with the energy of interaction being 17.6 kcal/mol less than that of the S,S-3a/PI complex. Although the absolute stereochemistry of individual enantiomers is not known, this study shows that R,R-3a interacts more favorably with PI than does S,S-3a and that (+)-3a is a more potent inhibitor of human platelet aggregation than (-)-3a. It is postulated that because of their lipophilicity, these compounds penetrate the platelet membrane and are then protonated at the pH of the cytosol. The protonated N then neutralizes the anionic charge on the membrane phosphoinositides, thereby rendering them less susceptible to hydrolysis by phospholipase C

  10. Sustained increase in platelet aggregation after the cessation of clopidogrel.

    PubMed

    Djukanovic, Nina; Todorovic, Zoran; Zamaklar-Trifunovic, Danijela; Protic, Dragana; Dzudovic, Boris; Ostojic, Miodrag; Obradovic, Slobodan

    2016-02-01

    This study shows that the abrupt cessation of one-year clopidogrel treatment was not associated with thrombotic events in a prospective, multicentre study that enrolled 200 patients subjected to coronary stent implantation and treated with aspirin + clopidogrel 1 year after the stent placement. The aim of the study was to investigate the causes of a sustained increase of platelet aggregability, considering that the values of platelet aggregation stimulated with ADP + PGE1 (ADPHS values) significantly increased 10-90 days after the cessation of clopidogrel. Values of platelet aggregation induced by thrombin receptor activating peptide (TRAP values) and arachidonic acid (ASPI values) were divided into quartiles on the basis of ADPHS values 10 days after stopping clopidogrel (ADPHS10 ). There was a significant difference between TRAP values divided into quartiles according to ADPHS10 , 10, 45 and 90 days after stopping clopidogrel (P < 0.001, all), and ASPI values across the same quartiles 10 and 45 days after the cessation of clopidogrel (P = 0.028 and 0.003). The results of the study indicate that patients with early pronounced rebound phenomena to clopidogrel termination have a long-term (at least 90 days) increased platelet aggregation to other agonists such as thrombin-related activated protein and arachidonic acid, suggesting the complex mutual relationship of various factors/agonists influencing the function of platelets.

  11. Effects of tomato extract on human platelet aggregation in vitro.

    PubMed

    Dutta-Roy, A K; Crosbie, L; Gordon, M J

    2001-06-01

    Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

  12. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated platelet aggregation system. 864.5700 Section 864.5700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology...

  13. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated platelet aggregation system. 864.5700 Section 864.5700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology...

  14. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated platelet aggregation system. 864.5700 Section 864.5700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology...

  15. 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

  16. Synthesis of huaicarbon A/B and their activating effects on platelet glycoprotein VI receptor to mediate collagen-induced platelet aggregation

    PubMed Central

    Yu, Hongli; Chen, Yeqing; Wu, Hao; Wang, Kuilong; Liu, Liping; Zhang, Xingde

    2017-01-01

    Quercetin and rhamnose were efficiently converted into huaicarbon A/B by heating at 250°C for 10-15 min or at 200°C for 25-30 min. With the optimum molar ratio of quercetin/rhamnose (1:3), huaicarbon A and B yields reached 25% and 16% respectively after heating at 250°C, with 55% quercetin conversion. Huaicarbon A/B both promoted washed platelet aggregation dose-dependently, which was antagonized by an inhibitor of glycoprotein VI (GPVI) receptor. Similarly, they both promoted collagen-induced platelet aggregation in platelet-rich plasma in dose-dependent manners. According to the S type dose-response model, EC50 values of huaicarbon A and huaicarbon B were calculated as 33.48 μM and 48.73 μM respectively. They induced intracellular Ca2+ accumulation that was specifically blocked by GPVI antagonist. Huaicarbon A/B enhanced intracellular Ca2+ accumulation and facilitated collagen-induced platelet aggregation, which were blocked by GPVI antagonist. They were conducive to collagen-induced platelet aggregation by activating platelet GPVI receptor. PMID:28337278

  17. Reduced platelet aggregation in a boy with scurvy.

    PubMed

    Dey, F; Möller, A; Kemkes-Matthes, B; Wilbrand, J-F; Krombach, G A; Neubauer, B; Hahn, A

    2012-11-01

    Pediatric scurvy is a rare condition characterized by perifollicular petechiae and bruising, hemorrhagic gingivitis and musculoskeletal symptoms, all assumed to be predominantly related to abnormal collagen structure. We report on a 9-year-old autistic boy with vitamin C deficiency due to a highly limited food range presenting with multiple petechiae, gum bleeding and debilitating bone pain, in whom platelet aggregometry revealed a distinctly reduced thrombocyte aggregation, normalizing after vitamin C supplementation. This observation indicates that platelet dysfunction may additionally contribute to the hemorrhagic diathesis in scurvy, and demonstrates that ascorbic acid deficiency should be considered in children with an otherwise unexplained acquired thrombocytopathy.

  18. Dose response of surfactants to attenuate gas embolism related platelet aggregation

    NASA Astrophysics Data System (ADS)

    Eckmann, David M.; Eckmann, Yonaton Y.; Tomczyk, Nancy

    2014-03-01

    Intravascular gas embolism promotes blood clot formation, cellular activation, and adhesion events, particularly with platelets. Populating the interface with surfactants is a chemical-based intervention to reduce injury from gas embolism. We studied platelet activation and platelet aggregation, prominent adverse responses to blood contact with bubbles. We examined dose-response relationships for two chemically distinct surfactants to attenuate the rise in platelet function stimulated by exposure to microbubbles. Significant reduction in platelet aggregation and platelet activation occurred with increasing concentration of the surfactants, indicating presence of a saturable system. A population balance model for platelet aggregation in the presence of embolism bubbles and surfactants was developed. Monte Carlo simulations for platelet aggregation were performed. Results agree qualitatively with experimental findings. Surfactant dose-dependent reductions in platelet activation and aggregation indicate inhibition of the gas/liquid interface's ability to stimulate cellular activation mechanically.

  19. Platelet aggregation in the formation of tumor metastasis

    PubMed Central

    Tsuruo, Takashi; Fujita, Naoya

    2008-01-01

    Metastasis is the major cause of death from cancer, yet the optimal strategy against it remains uncertain. The pathogenesis of hematogenous metastasis is dynamic and consists of the following steps: 1) detachment of tumor cells from the primary site, 2) invasion into the host’s blood vessels, 3) migration in the host’s blood stream, 4) transport along the circulation, 5) arrest in or adhesion to the capillary in a distant organ, 6) extravasation, and 7) proliferation within the foreign tissues. A key to successful hematogenous metastasis is tumor survival in the bloodstream because most circulating tumor cells are rapidly destroyed by the shear forces or are attacked by the immune system. Less than 0.01% of these cells result in metastasis. Tumor cell–induced platelet aggregation has been reported to facilitate hematogenous metastasis by increasing the arrest of tumor cell emboli in the microcirculation. Platelet aggregation is also believed to protect tumor cells from immunological assault in the circulation. We have identified Aggrus as a platelet–aggregating factor expressed on a number of human cancers. Because hematogenous metastasis is reduced when neutralizing antibodies or eliminating carbohydrates attenuates Aggrus function, Aggrus’s main contribution to hematogenous metastasis of Aggrus–expressing cells, then, is by promoting platelet aggregation. Aggrus could serve as an ideal target for drug development to block metastasis. PMID:18941298

  20. Effects of argon laser on in vitro aggregation of platelets in platelet rich plasma and whole blood

    SciTech Connect

    Doerger, P.T.; Glueck, H.I.; McGill, M.

    1988-06-01

    The effects of an Argon laser on platelet aggregation were studied, since platelets may be exposed to laser energy when used intravascularly. Various preparations of platelets in platelet rich plasma (PRP) and whole blood, with or without aspirin, were tested with the aggregating agents ADP, collagen, thrombin, and epinephrine. Simultaneous release of ATP was also measured in PRP. At relatively low levels of irradiation, platelet aggregation was potentiated. Enhancement was evidenced by an increase in percent aggregation, earlier onset of the reaction, and reduction in the amount of aggregating agent required. In PRP, the mechanism of laser potentiation appeared to be the release of endogenous ATP from platelets. At relatively high levels of irradiation, platelets were destroyed and aggregation abolished. In whole blood, the mechanism was somewhat more complicated since release of ATP occurred from RBCs as well as platelets. Spontaneous aggregation following laser treatment occurred in isolated instances in PRP and in every trial in whole blood preparations. Aspirin ingestion inhibited the laser's effects in PRP but not in whole blood. These results may have important clinical implications for laser angioplasty, and the potentiated aggregation response may prove useful in laboratory studies of platelet function.

  1. [Mean platelet volume: interactions with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels].

    PubMed

    Khaspekova, S G; Ziuriaev, I T; Iakushkin, V V; Naĭmushin, Ia A; Sirotkina, O V; Zaĭtseva, N O; Ruda, M Ia; Mazurov, A V

    2014-01-01

    Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) were investigated in this study. Investigation was performed in a group of healthy volunteers (n = 38) and in a group of patients with acute coronary syndrome (ACS). In patients blood was collected at days 1, 3-5 and 8-12 after ACS development. As an antiaggregant therapy all patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) and most of them--clopidogrel (ADP receptor antagonist) with the exception of part of the patients (n = 44) at day 1 who had not taken clopidogrel before first blood collection. In volunteers platelet aggregation was stimulated by 1.25, 2.5, 5 and 20 M ADP, and in patients--by 5 and 20 M ADP. GP IIb-IIIa and GP Ib content on platelet surface was measured using 125I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels were revealed at 1.25 and 2.5 M ADP (coefficients of correlation (r)--0.396 and 0.373, p < 0.05) and at 5 and 20 those interactions did not reach significant level (r--0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients who received ASA but had not started clopidogrel treatment (r--0.526, p < 0.01 and 0.368, p < 0.05 for 5 and 20 M ADP respectively). Interactions between these parameters were not registered upon combined treatment with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in healthy donors and ACS patients (at all time points) -r from 0.439 to 0.647 (p < or = 0.001 for all correlations). Genetic

  2. The automation of routine light transmission platelet aggregation

    PubMed Central

    Lawrie, A S; Kobayashi, K; Lane, P J; Mackie, I J; Machin, S J

    2014-01-01

    Introduction The investigation of platelet function by aggregometry requires specialist equipment and is labour intensive. We have developed an automated platelet aggregation method on a routine coagulation analyser. Methods We used a CS-2000i (Sysmex) with prototype software to perform aggregation in platelet-rich plasma (PRP), using the following agonists: ADP (0.5–10 μm), epinephrine (0.5–10 μm), collagen (0.5–10 mg/μL), ristocetin (0.75–1.25 mg/mL) and arachidonic acid (0.12–1.0 mm). Platelet agonists were from Hyphen Biomed, and an AggRAM aggregometer (Helena Biosciences) was used as the reference instrument. Results CS-2000i reaction cuvette stirrer speed was found to influence reaction sensitivity and was optimized to 800 rpm. There were no clinically significant changes in aggregation response when the PRP platelet count was 150–480 x 109/L, but below this there were changes in the maximum amplitude (MA) and slope (rate). Dose response with each of the agonists was comparable between CS-2000i and an AggRAM aggregometer and normal subjects receiving antiplatelet drugs. Aggregation imprecision was similar on both the CS-2000i and AggRAM systems, with a cv for 2–5 μm ADP MA and slope varying between 3–12%. Conclusion Our preliminary studies indicated that optimal sensitivity using the CS-2000i was obtained with a reaction cuvette stirrer speed of 800 rpm and a PRP platelet count of 200–300 x 109/L; aggregation with a PRP count <100 x 109/L showed poor sensitivity. Imprecision and detection of antiplatelet drug effects was similar between the CS-2000i and AggRAM. These data demonstrate that CS-2000i is comparable to a stand-alone aggregometer, although CS-2000i has the advantages of walk-away technology and also required a smaller sample volume than the AggRAM (44% less). PMID:24237750

  3. Platelet anti-aggregation activities of compounds from Cinnamomum cassia.

    PubMed

    Kim, Sun Young; Koo, Yean Kyoung; Koo, Ja Yong; Ngoc, Tran Minh; Kang, Sam Sik; Bae, KiHwan; Kim, Yeong Sik; Yun-Choi, Hye Sook

    2010-10-01

    Cinnamomum cassia is a well-known traditional medicine for improvement of blood circulation. An extract of this plant showed both platelet anti-aggregation and blood anti-coagulation effects in preliminary testing. Among the 13 compounds obtained from this plant, eugenol (2), amygdalactone (4), cinnamic alcohol (5), 2-hydroxycinnamaldehyde (7), 2-methoxycinnamaldehyde (8), and coniferaldehyde (9) showed 1.5-73-fold greater inhibitory effects than acetylsalicylic acid (ASA) on arachidonic acid (AA)-induced aggregation (50% inhibitory concentration [IC₅₀] = 3.8, 5.16, 31.2, 40.0, 16.9, and 0.82 μM, respectively, vs. 60.3 μM) and 6.3-730-fold stronger effect than ASA on U46619 (a thromboxane A₂ mimic)-induced aggregation (IC₅₀ = 3.51, 33.9, 31.0, 51.3, 14.6, and 0.44 μM, respectively, vs. 321 μM). The other compounds, coumarin (3), cinnamaldehyde (6), cinnamic acid (10), icariside DC (11), and dihydrocinnacasside (12), also inhibited (2.5 to four times greater than ASA) U46619-induced aggregation. In addition, compounds 2, 4, 5, 6, 7, 8, and 9 were 1.3-87 times more effective than ASA against epinephrine-induced aggregation (IC₅₀ = 1.86, 1.10, 37.7, 25.0, 16.8, 15.3, and 0.57 μM, respectively, vs. 50.0 μM). However, the 13 compounds were only very mildly effective against blood coagulation, if at all. In conclusion, compounds 2, 4, 8, and 9 showed stronger inhibitory potencies than others on AA-, U46619-, and epinephrine-induced platelet aggregation. Eugenol (2) and coniferaldehyde (9) were the two of the most active anti-platelet constituents of C. cassia.

  4. Platelet anti-aggregant property of some Moroccan medicinal plants.

    PubMed

    Mekhfi, Hassane; El Haouari, Mohammed; Legssyer, Abdelkhaleq; Bnouham, Mohammed; Aziz, Mohammed; Atmani, Fouad; Remmal, Adnane; Ziyyat, Abderrahim

    2004-10-01

    It is known that blood platelets may present some dysfunction linked to cardiovascular pathologies such as arterial hypertension. The aim of this work is to examine the in vitro anti-aggregant effect of five medicinal plants among which three were reported as antihypertensive in oriental Morocco: Arbutus unedo (Ericaceae), Urtica dioïca (Urticaceae), and Petroselinum crispum (Apiaceae). The two other plants were Cistus ladaniferus (Cistaceae) and Equisetum arvense (Equisetaceae). The results obtained showed that all extracts produced a dose-dependent inhibition of thrombin and ADP-induced aggregation. The calculated IC50 (half-maximal inhibition of thrombin and ADP-induced aggregation) was found to be identical in all plant extracts while Urtica dioïca had a higher IC50 value. The effect of plants could be related in part to the polyphenolic compounds present in their extracts suggesting their involvement in the treatment or prevention of platelet aggregation complications linked to cardiovascular diseases. Phytochemical separation must be carried out to identify the active principles responsible for the anti-aggregant effect and elucidate their mechanisms of action.

  5. Abnormal platelet aggregation in patients with Raynaud's phenomenon.

    PubMed Central

    Biondi, M L; Marasini, B

    1989-01-01

    Platelet aggregation in vitro to several aggregating agents (serotonin (5-HT), adenosine diphosphate, adrenaline and collagen) was studied in 16 patients with primary and secondary Raynaud's phenomenon and compared with that in 13 normal volunteers. Platelets from patients with Raynaud's phenomenon had significantly greater responses to all the 5-HT concentrations tested (p less than 0.001 for 10 microM; p less than 0.01 for 1 microM; p less than 0.05 for 0.1 microM; p less than 0.02 for 0.025 microM) and to low doses of adenosine diphosphate (p less than 0.01 for 1 microM; p less than 0.02 for 0.5 microM) but normal responses to collagen, adrenaline, and high doses of adenosine diphosphate. Patients with secondary Raynaud's phenomenon were significantly more hypersensitive to 0.5 microM adenosine diphosphate than patients with primary Raynaud's phenomenon. In patients with secondary Raynaud's phenomenon there was a significant correlation between the extent of 5-HT aggregation and the duration of the disease. The finding that platelets from patients with Raynaud's phenomenon have enhanced responses to 5-HT and adenosine diphosphate, but normal responses to adrenaline and collagen, is consistent with a role for 5-HT in this disease. PMID:2760232

  6. The effect of urethane and thiopental sodium on platelet aggregation in vitro and in vivo.

    PubMed

    Evangelista, S; Abelli, L; Maggi, C A; Meli, A

    1984-09-01

    The potential in vitro (heparinized or citrated PRP) and in vivo effects of urethane and thiopental sodium on arachidonic acid, collagen, or ADP-induced rat platelet aggregation has been investigated. Both anesthetics antagonized platelet aggregation in vitro at concentrations higher than those found in plasma during anesthesia. Neither anesthetic altered the piastrinopenia induced by intravenous administration of these aggregating agents. These findings suggest that both anesthetics are suitable for in vivo platelet aggregation studies.

  7. Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

    PubMed Central

    Boudejltia, Karim Zouaoui; Ribeiro de Sousa, Daniel; Uzureau, Pierrick; Yourassowsky, Catherine; Perez-Morga, David; Courbebaisse, Guy; Chopard, Bastien; Dubois, Frank

    2015-01-01

    Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

  8. In vitro effects of ethanol on the pathways of platelet aggregation

    SciTech Connect

    Rand, M.L.; Kinlough-Rathbone, R.L.; Packham, M.A.; Mustard, J.F.

    1986-03-01

    Ethanol is reported to inhibit platelet aggregation in vivo and in vitro, but the mechanisms of its action on stimulus-response coupling in platelets is unknown. Platelet aggregation to thrombin occurs through at least three pathways: released ADP; thromboxane A/sub 2/ (TXA/sub 2/); and a third pathway(s). Aggregation of rabbit platelets in citrated platelet-rich plasma (PRP) or washed suspensions to ADP (0.5-10 ..mu..M) was not affected by ethanol, at concentrations up to 5 mg/ml (lethal). Primary ADP-induced (5 ..mu..M) aggregation of human platelets in PRP was also unaffected by ethanol, but secondary aggregation and release of /sup 14/C-serotonin, due to TXA/sub 2/ formation, was inhibited by ethanol (2 and 4 mg/ml). Since arachidonate (AA)-induced (25-250 ..mu..M) aggregation and release by washed rabbit platelets was unaltered by ethanol, it may inhibit mobilization of AA from platelet membrane phospholipids. Ethanol (2-4 mg/ml) inhibited rabbit platelet aggregation and release to low concentrations of thrombin (< 10 mU/ml) or collagen, and also inhibited aggregation and release of aspirin-treated (500 ..mu.. M) rabbit platelets (that cannot form TXA/sub 2/) to low concentrations of thrombin (< 10 mU/ml). Thus, ethanol does not inhibit the mobilization of AA, and partially inhibits the third pathway(s) of platelet aggregation.

  9. Characterization of UBO-QIC as a Gαq inhibitor in platelets.

    PubMed

    Inamdar, Vaishali; Patel, Akruti; Manne, Bhanu Kanth; Dangelmaier, Carol; Kunapuli, Satya P

    2015-01-01

    Gαq plays an important role in platelet activation by agonists such as thrombin, adenosine diphosphate (ADP) and thromboxane. The significance of Gαq signaling in platelets was established using YM254890, a Gαq/11-specific inhibitor and Gαq knockout murine platelets. However, YM-254890 is no longer available for investigators and there is a need to characterize other Gαq inhibitors. The aim of this study is to characterize the specificity of a compound, {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4-methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7 → 1)-lactone (9CI)} (UBO-QIC), as a Gαq inhibitor in platelets. Human platelets treated with UBO-QIC showed a concentration-dependent inhibition of platelet aggregation and secretion by protease-activated receptors (PAR) agonists, U46619 and ADP. UBO-QIC also abolished Gαq pathway signaling events such as calcium mobilization and pleckstrin phosphorylation. UBO-QIC had no nonspecific effects on the Gα12/13 pathway since platelet shape change was intact in Gαq knockout murine platelets stimulated with PAR agonists in the presence of the inhibitor. In addition, UBO-QIC-treated platelets did not affect collagen-related peptide-induced platelet activation suggesting that this inhibitor had no non-specific effects on the GPVI pathway. Furthermore, Akt phosphorylation downstream of the Gαi and Gαz pathways, and vasodilator-stimulated phosphoprotein phosphorylation downstream of the Gαs pathway were not inhibited in UBO-QIC-treated platelets. UBO-QIC is a specific inhibitor for Gαq, which can be a useful tool for investigating Gαq-coupled receptor signaling pathways in platelets.

  10. Platelet Integrin αIIbβ3 Inhibitor Rescues Progression of Apoptosis in Human Platelets

    PubMed Central

    Zhu, Jie; Wang, Qinghang; Nie, Yumei; Yan, Rong; Dai, Kesheng; Zhou, Birong

    2016-01-01

    Background Apoptosis plays an important role in the physiology of platelet function. We aimed to detect the effect of the platelet integrin αIIbβ3 inhibitor, tirofiban, on apoptotic events, including mitochondrial inner-membrane potential (ΔΨm), phosphatidylserine (PS) exposure on platelet surface, and the generation of reactive oxygen species (ROS), when washed platelets were stimulated with thrombin. Material/Methods The study included washed platelets from healthy humans, divided into 4 groups: vehicle, and tirofiban (0.05 μg/ml, 0.25 μg/ml, and 0.5 μg/ml). Platelets were pretreated with vehicle or tirofiban and incubated at 37°C with agitation for 6 h and 24 h. Before thrombin addition, the vehicle group divided into 2 equal groups. Except one vehicle group, the other 4 groups were all stimulated with thrombin (1 U/ml) for 30 min at 37°C. Using flow cytometry, we studied the ΔΨm and PS exposure on platelet surfaces, and the generation of ROS in platelets. Results We observed that at the time of 6 h and 24 h, thrombin-stimulated vehicle platelets induced significant depolarization of ΔΨm, higher PS exposure, and increased ROS production compared with the vehicle group (P<0.01). However, the tirofiban group had significantly more recovery of ΔΨm, PS exposure, and ROS production compared with the thrombin group (P<0.01). Conclusions The platelet integrin αIIbβ3 inhibitor, tirofiban, inhibits the depolarization of ΔΨm, PS exposure on platelet surface, and ROS production when stimulated with thrombin. These results suggest that αIIbβ3 inhibitor inhibits the initiation of apoptosis in platelets, showing a potential clinical application of tirofiban as an apoptosis inhibitor. PMID:27827357

  11. Platelet-adenovirus vs. inert particles interaction: effect on aggregation and the role of platelet membrane receptors.

    PubMed

    Gupalo, Elena; Kuk, Cynthia; Qadura, Mohammad; Buriachkovskaia, Liudmila; Othman, Maha

    2013-01-01

    Platelets are involved in host defense via clearance of bacteria from the circulation, interaction with virus particles, and uptake of various size particulates. There is a growing interest in micro- and nanoparticles for drug delivery and there is evidence that the properties of these particles critically influence their interaction and uptake by various tissues and cells including platelets. Virus mediated gene therapy applications are still challenged by the resultant thrombocytopenia and the mechanism(s) of platelet-foreign particles interaction remains unclear. We studied the specifics of platelet interaction with an active biological agent (adenovirus) and inert latex microspheres (MS) and investigated the role of platelet proteins in this interaction. We show that activated and not resting platelets internalize MS, without influencing platelet aggregation. In contrast, adenovirus induces and potentiates ADP-induced platelet aggregation and results in rapid expression of P-selectin. Platelets then internalize adenovirus and viral particles appear inside the open canalicular system. Inhibition of platelet αIIbβ3, GPIbα, and P-selectin decreases both platelet aggregation and internalization of MS. Inhibition of αIIbβ3 and αVβ3 does not abolish adenovirus platelet internalization and adenovirus-induced platelet activation is maintained. Our study demonstrates that platelets react differentially with foreign particles and that αIIbβ3 is a key player in platelet engulfing of foreign particles but not in mediating adenovirus internalization. Other platelet candidate molecules remain to be investigated as potential targets for management of adenovirus-induced thrombocytopenia.

  12. Inhibition of glutamate receptors reduces the homocysteine-induced whole blood platelet aggregation but does not affect superoxide anion generation or platelet membrane fluidization.

    PubMed

    Karolczak, Kamil; Pieniazek, Anna; Watala, Cezary

    2017-01-01

    Homocysteine (Hcy) is an excitotoxic amino acid. It is potentially possible to prevent Hcy-induced toxicity, including haemostatic impairments, by antagonizing glutaminergic receptors. Using impedance aggregometry with arachidonate and collagen as platelet agonists, we tested whether the blockade of platelet NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors with their inhibitors: MK-801 (dizocilpine hydrogen maleate, [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), CNQX (7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile) and UBP-302 (2-{[3-[(2S)-2-amino-2-carboxyethyl]-2,6-dioxo-3,6-dihydropyrimidin 1(2H)-yl]methyl}benzoic acid) may hamper Hcy-dependent platelet aggregation. All the tested compounds significantly inhibited Hcy-augmented aggregation of blood platelets stimulated either with arachidonate or collagen. Hcy stimulated the generation of superoxide anion in whole blood samples in a concentration-dependent manner; however, this process appeared as independent on ionotropic glutamate receptors, as well as on NADPH oxidase and protein kinase C, and was not apparently associated with the extent of either arachidonate- or collagen-dependent platelet aggregation. Moreover, Hcy acted as a significant fluidizer of surface (more hydrophilic) and inner (more hydrophobic) regions of platelet membrane lipid bilayer, when used at the concentration range from 10 to 50 µmol/l. However, this effect was independent on the Hcy action through glutamate ionotropic receptors, since there was no effects of MK-801, CNQX or UBP-302 on Hcy-mediated membrane fluidization. In conclusion, Hcy-induced changes in whole blood platelet aggregation are mediated through the ionotopic excitotoxic receptors, although the detailed mechanisms underlying such interactions remain to be elucidated.

  13. Platelets aggregation in pathological conditions: role of local shear rates and platelet activation delay time.

    NASA Astrophysics Data System (ADS)

    Li, He; Zarif Khalili Yazdani, Alireza; Karniadakis, George

    2015-11-01

    Platelets play an essential role in the initiation and formation of a thrombus, however their detailed motion in blood vessels with complex geometries, such as in the aneurysmal vessel or stenotic vessel in atherosclerosis, has not been studied systematically. Here, we perform spectral element simulations (NEKTAR code) to obtain the 3D flow field in blood vessel with cavities, and we apply the force coupling method (FCM) to simulate the motion of platelets in blood flow. Specifically, simulations of platelets are performed in a 0.25 mm diameter circular blood vessel with 1 mm length. Corresponding coarse-grained molecular dynamics simulations are employed to provide input to the NEKTAR-FCM code. Simulations are conducted at several different Reynolds numbers (Re). An ellipsoid-shaped cavity is selected to intersect with the middle part of the circular vessel to represent the aneurysmal part of the blood vessel. Based on the simulation results, we quantify how the platelets motion and aggregation in the blood vessel cavities depend on Re, platelet activation delay time, and the geometry of the cavities.

  14. [Effect of magnesium, acetylsalicilic acid and emoxypine on aggregation of platelets].

    PubMed

    Loznikova, S Zh; Sukhodola, A A; Shcharbina, N Iu; Shcharbin, D G

    2014-01-01

    The thrombin-induced platelet aggregation was studied in the absence and presence of magnesium sulfate, acetylsalicylic acid and emoxypine. It was found that all the preparations studied were able separately to decrease platelet aggregation. In contrast, their joint action was not able to affect the aggregation of platelets. The data obtained can be used to choose the treatment strategy for patients with ischemic stroke.

  15. Effect of the Garlic Pill in comparison with Plavix on Platelet Aggregation and Bleeding Time

    PubMed Central

    Fakhar, H; Hashemi Tayer, A

    2012-01-01

    Introduction Platelet aggregation plays a significant role in the etiology of cardiovascular diseases. Therefore, treatments to inhibit platelet aggregation can reduce the risk of coronary thrombosis. Several studies indicated that garlic can inhibit platelet aggregation. This study aimed to determine the effect of garlic in comparison with Plavix on platelet aggregation. Materials and Methods In this randomized clinical trial, platelet aggregation and bleeding time was obtained from 36 healthy volunteers. Volunteers were randomly divided into 4 groups. The first, second and third groups respectively received 600, 1200 and 2400 mg garlic and the fourth group received 75 mg Plavix for three weeks. Afterwards, platelet aggregation and bleeding time were both evaluated and the results before and after the study were compared. Data were analyzed using SPSS software version 16. Results Platelet aggregation induced by adenosine diphosphate and arachidonic acid agonists decreased in the groups that used 1200 or 2400 mg garlic. This difference was statistically significant (p<0.05). As compared to the other groups, the bleeding time also increased in those received 2400 mg garlic pill. Conclusion Since garlic can inhibit platelet aggregation, it is suggested to use it as a supplementary treatment to reduce platelet aggregation is highly recommended. PMID:24575255

  16. Imperanene, a novel phenolic compound with platelet aggregation inhibitory activity from Imperata cylindrica.

    PubMed

    Matsunaga, K; Shibuya, M; Ohizumi, Y

    1995-01-01

    Imperanene, a novel phenolic compound [1] has been isolated from Imperata cylindrica. Its structure was elucidated by spectroscopic evidence. Imperanene showed platelet aggregation inhibitory activity.

  17. Platelet 5-hydroxytryptamine release and aggregation promoted by cotton bracts tannin.

    PubMed

    Rohrbach, M S; Rolstad, R A; Tracy, P B; Russell, J A

    1984-01-01

    The effect of aqueous extracts of cotton bract (CBE) on platelet secretion and aggregation was examined by using washed bovine and human platelets. The CBE promoted the release of 75% to 90% of the 5-hydroxytryptamine (5-HT) stored in both human and bovine platelets in a dose-dependent manner. This release reaction occurred without the lysis of the platelets and was not inhibited by indomethacin, 2-deoxyglucose, or KCN. Fractionation of the CBE indicated that the platelet secretagogue present in the CBE was the condensed polyphenol, tannin. In addition to promoting the secretion of 5-HT, tannin also aggregated the platelets in a dose-dependent manner. We conclude that the secretion of platelet 5-HT and the aggregation of platelets by tannin could potentially contribute to the pulmonary symptoms associated with byssinosis.

  18. L-4F Alters Hyperlipidemic (but not Normal) Mouse Plasma to Reduce Platelet Aggregation

    PubMed Central

    Buga, Georgette M.; Navab, Mohamad; Imaizumi, Satoshi; Reddy, Srinivasa T.; Yekta, Babak; Hough, Greg; Chanslor, Shawn; Anantharamaiah, G.M.; Fogelman, Alan M.

    2010-01-01

    Objective Hyperlipidemia is associated with platelet hyper-reactivity. We hypothesized that L-4F, an apoA-I mimetic peptide, would inhibit platelet aggregation in hyperlipidemic mice. Methods and Results Injecting L-4F into apoE null and LDL receptor null mice resulted in a significant reduction in platelet aggregation in response to agonists but there was no reduction in platelet aggregation after injection of L-4F into wild-type (WT) mice. Consistent with these results, injection of L-4F into apoE null mice prolonged bleeding time but not in WT mice. Incubating L-4F in vitro with apoE null platelet rich plasma also resulted in decreased platelet aggregation. However, incubating washed platelets from either apoE null or WT mice with L-4F did not alter aggregation. Compared to wild-type mice, unstimulated platelets from apoE null mice contained significantly more 12-HETE, thromboxane A2 (TXA2), prostaglandins D2 (PGD2) and E2 (PGE2). In response to agonists, platelets from L-4F treated apoE null mice formed significantly less TXA2, PGD2 PGE2, and 12-HETE. Conclusions By binding plasma oxidized lipids that cause platelet hyper-reactivity in hyperlipidemic mice, L-4F decreases platelet aggregation. PMID:19965777

  19. Tau protein and tau aggregation inhibitors.

    PubMed

    Bulic, Bruno; Pickhardt, Marcus; Mandelkow, Eva-Maria; Mandelkow, Eckhard

    2010-01-01

    Alzheimer disease is characterized by pathological aggregation of two proteins, tau and Abeta-amyloid, both of which are considered to be toxic to neurons. In this review we summarize recent advances on small molecule inhibitors of protein aggregation with emphasis on tau, with activities mediated by the direct interference of self-assembly. The inhibitors can be clustered in several compound classes according to their chemical structure, with subsequent description of the structure-activity relationships, showing that hydrophobic interactions are prevailing. The description is extended to the pharmacological profile of the compounds in order to evaluate their drug-likeness, with special attention to toxicity and bioavailability. The collected data indicate that following the improvements of the in vitro inhibitory potencies, the consideration of the in vivo pharmacokinetics is an absolute prerequisite for the development of compounds suitable for a transfer from bench to bedside.

  20. Selective deficiency in collagen-induced platelet aggregation during L-asparaginase therapy.

    PubMed

    Shapiro, R S; Gerrard, J M; Ramsay, N K; Nesbit, M E; Coccia, P F; Stoddard, S F; Plow, E F; White, J G; Krivit, W

    1980-01-01

    Platelet aggregation studies were performed on 10 pediatric patients with acute lymphoblastic leukemia (ALL) receiving induction therapy with vincristine, prednisone, and L-asparaginase. An isolated abnormality in platelet aggregation in response to collagen was found in all patients during the course of therapy. Platelet aggregation in response to collagen normalized following the discontinuation of L-asparaginase, while patients were still on vincristine and prednisone. In contrast to the abnormal collagen response, platelet aggregation induced by epinephrine, arachidonic acid, adenosine diphosphate (ADP), and thrombin were normal both during and following therapy. In the one patient with a normal platelet count before therapy, aggregation induced by all agents was normal. This selective abnormality in collagen aggregation therefore appears to result from therapy, with the use of L-asparaginase in particular being implicated.

  1. Procoagulant platelets form an α-granule protein-covered "cap" on their surface that promotes their attachment to aggregates.

    PubMed

    Abaeva, Anastasia A; Canault, Matthias; Kotova, Yana N; Obydennyy, Sergey I; Yakimenko, Alena O; Podoplelova, Nadezhda A; Kolyadko, Vladimir N; Chambost, Herve; Mazurov, Aleksei V; Ataullakhanov, Fazoil I; Nurden, Alan T; Alessi, Marie-Christine; Panteleev, Mikhail A

    2013-10-11

    Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbβ3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbβ3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.

  2. Platelet–cancer interactions: mechanisms and pharmacology of tumour cell-induced platelet aggregation

    PubMed Central

    Jurasz, Paul; Alonso-Escolano, David; Radomski, Marek W

    2004-01-01

    During haematogenous metastasis, cancer cells migrate to the vasculature and interact with platelets resulting in tumour cell-induced platelet aggregation (TCIPA). We review: The biological and clinical significance of TCIPA; Molecular mechanisms involved in platelet aggregation by cancer cells; Strategies for pharmacological regulation of these interactions. We conclude that pharmacological regulation of platelet–cancer cell interactions may reduce the impact of TCIPA on cancer biology. PMID:15492016

  3. In vitro effects of mercury on platelet aggregation, thromboxane and vascular prostacyclin production.

    PubMed

    Caprino, L; Togna, A R; Cebo, B; Dolci, N; Togna, G

    1983-01-01

    Mercuric chloride [25-50 micrograms/ml platelet-rich plasma (PRP)] lowers the threshold concentration of arachidonic acid (AA) required for triggering rabbit platelet aggregation and causes a marked increase of thromboxane production. The metal, added as HgCl2, does not modify (50 micrograms/ml PRP) or block (100 micrograms/ml PRP) the platelet aggregation wave induced by a normal aggregating dose of AA. Whether or not AA-induced platelet aggregation takes place, a large increase in thromboxane production is observed. Methyl mercury, assayed as reference drug, induces platelet aggregation and a significant increase of thromboxane levels. Finally, HgCl2 and methyl mercury, in a concentration range of 0.125-0.5 micrograms/microliters in the incubation liquid, induce an increased prostacyclin release from rat aortic tissue.

  4. [Mechanism of action of biogenic chloramines and hypochlorite on initial aggregation of blood platelets].

    PubMed

    Murina, M A; Savel'eva, E L; Roshchupkin, D I

    2006-01-01

    The antiaggregant action of two reactive oxidants N,N-dichlorotaurine (chloramine of biogenic type) and sodium hypochlorite on the initial ADP-induced aggregation of rabbit blood platelets has been studied. Platelet aggregation in the reconstructed platelet-rich plasma (PRP) was measured by the nephelometric method, and the aggregation index was an increase in the intensity of small-angle light scattering. The introduction of chloramine at comparatively small concentrations (no greater than 1 mM active chlorine) directly into the reconstructed platelet-rich plasma induces the suppression of the initial aggregation (formation of small aggregates) several times stronger than in the case of its preliminary incubation with plasma alone. This suggests that N,N-dichlorotaurine exerts its antiaggeregant action on the platelet-rich plasma by direct interaction with cells. The effects of the inhibition of platelet aggregation in two variants of introduction of high concentrations of N,N-dichlorotaurine do not significantly differ. In this case a great amount of residual chloramine remains in the plasma, which just induces the suppression of platelet aggregation during subsequent reconstruction of the platelet-rich plasma. Similar data have been obtained in the study of the antiaggregant action of hypochlorite. N,N-Dichlorotaurine and hypochlorite at final concentrations of 0.2-0.3 and 0.15 mM, respectively, inhibit strongly the initial aggregation of isolated platelets (approximately 2 x 10(8) cells in 1 ml) preliminarily activated for 1.5 min by the addition of 100-500 nM ADP. However, the antiaggregants show a more profound suppression of aggregation of unstimulated platelets. The antiaggregant effects of N,N-dichlorotaurine and hypochlorite are probably due to the oxidative modification of sulfur-containing groups in platelet plasmatic membrane.

  5. Inhalation of Whole Diesel Exhaust but not Gas-Phase Components Affects In Vitro Platelet Aggregation in Hypertensive Rats

    EPA Science Inventory

    Rationale: Intravascular thrombosis and platelet aggregation are enhanced following exposure to diesel exhaust (DE) and other respirable particulate matter; however, the roles of endothelial and circulating mediators on platelet aggregation remain unclear. We hypothesized that ad...

  6. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

    PubMed

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

    2009-08-17

    Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

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

    PubMed

    Matsuhisa, Fumikazu; Kitamura, Nobuo; Satoh, Eiki

    2014-03-01

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

  8. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    NASA Astrophysics Data System (ADS)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-07-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

  9. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation

    PubMed Central

    Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation. PMID:28072855

  10. SDF-1α/CXCR4 Signaling in Lipid Rafts Induces Platelet Aggregation via PI3 Kinase-Dependent Akt Phosphorylation.

    PubMed

    Ohtsuka, Hiroko; Iguchi, Tomohiro; Hayashi, Moyuru; Kaneda, Mizuho; Iida, Kazuko; Shimonaka, Motoyuki; Hara, Takahiko; Arai, Morio; Koike, Yuichi; Yamamoto, Naomasa; Kasahara, Kohji

    2017-01-01

    Stromal cell-derived factor-1α (SDF-1α)-induced platelet aggregation is mediated through its G protein-coupled receptor CXCR4 and phosphatidylinositol 3 kinase (PI3K). Here, we demonstrate that SDF-1α induces phosphorylation of Akt at Thr308 and Ser473 in human platelets. SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the CXCR4 antagonist AMD3100 or the PI3K inhibitor LY294002. SDF-1α also induces the phosphorylation of PDK1 at Ser241 (an upstream activator of Akt), GSK3β at Ser9 (a downstream substrate of Akt), and myosin light chain at Ser19 (a downstream element of the Akt signaling pathway). SDF-1α-induced platelet aggregation is inhibited by pretreatment with the Akt inhibitor MK-2206 in a dose-dependent manner. Furthermore, SDF-1α-induced platelet aggregation and Akt phosphorylation are inhibited by pretreatment with the raft-disrupting agent methyl-β-cyclodextrin. Sucrose density gradient analysis shows that 35% of CXCR4, 93% of the heterotrimeric G proteins Gαi-1, 91% of Gαi-2, 50% of Gβ and 4.0% of PI3Kβ, and 4.5% of Akt2 are localized in the detergent-resistant membrane raft fraction. These findings suggest that SDF-1α/CXCR4 signaling in lipid rafts induces platelet aggregation via PI3K-dependent Akt phosphorylation.

  11. [Effect of dauricine on rat and human platelet aggregation and metabolism of arachidonic acid in washed rat platelets].

    PubMed

    Tong, L; Yue, T L

    1989-01-01

    Dauricine (Dau), an isoquinoline alkaloid extracted from the roots of Menispermum dauricum D. C. and used as an antiarrhythmic agent in China recently, was shown to inhibit rat platelet aggregation induced by arachidonic acid (AA) and ADP, as well as human platelet aggregation induced by AA, ADP and adrenaline (Adr) in vitro in a dose-dependent manner. The concentration of Dau required for 50% inhibition (IC50) of rat platelet aggregation induced by AA and ADP was 26 and 37 mumol/L, respectively. For human platelet aggregation induced by AA, ADP and Adr the IC50 of Dau was found to be 39, 55 and 43 mumol/L, respectively. Dau inhibited the cyclooxygenase pathway metabolites of AA (TXB2 and HHT) in washed intact rat platelets. The production of TXB2 and HHT was reduced by 26% and 19%, respectively, when the Dau concentration was 50 mumol/L and by 46 and 45%, respectively, when the concentration of Dau was 100 mumol/L. The formation of 12-HETE was also inhibited at 100 mumol/L of Dau. The inhibitory effect of Dau on AA metabolism may be one of the mechanisms related to its inhibition of platelet aggregation.

  12. Role of red cells in preventing the growth of platelet aggregation.

    PubMed

    Machi, J; Sigel, B; Ramos, J R; Justin, J R; Feinberg, H; LeBreton, G C; Robertson, A L

    1984-10-01

    Using high-resolution real-time two-dimensional ultrasound, we have investigated the role of red cells in the growth of already established platelet aggregates under controlled flow conditions. Platelet rich plasma (PRP) was circulated in vitro in horizontally and vertically arranged tubing at mean shear rate ranging from 60 to 0 sec-1, and adenosine diphosphate (ADP) was used to induce platelet aggregation. ADP-induced platelet aggregates grew in size and tended to sediment as shear rate decreased, in particular, below 10 sec-1. At 0 sec-1 (stasis), large clusters of platelet aggregates formed. The addition of washed red cells to produce a hematocrit of only 2% significantly interfered with the growth and sedimentation of platelet aggregates as shear rate was reduced. Formaldehyde-hardened erythrocytes had a similar effect in preventing the growth of platelet aggregates, suggesting that mechanical collision of red cells with platelet aggregates may be the cause of growth inhibition. Therefore, the thrombotic process may be enhanced in red cell poor zones in circulation resulting from flow disturbances associated with vascular stenosis or within artificial organs and extracorporeal systems. The present study also suggested that red cell free PRP should be carefully administered therapeutically.

  13. Effect of coffee drinking on platelets: inhibition of aggregation and phenols incorporation.

    PubMed

    Natella, F; Nardini, M; Belelli, F; Pignatelli, P; Di Santo, S; Ghiselli, A; Violi, F; Scaccini, C

    2008-12-01

    Epidemiological studies indicate a J-shaped relationship linking coffee consumption and cardiovascular risk, suggesting that moderate coffee consumption can be beneficial. Platelet aggregation is of critical importance in thrombotic events, and platelets play a major role in the aetiology of several CVD. The aim of this study was to evaluate the effect of coffee drinking on platelet aggregation ex vivo, using caffeine as control. A crossover study was performed on ten healthy subjects. In two different sessions, subjects drank 200 ml coffee, containing 180 mg caffeine, or a capsule of caffeine (180 mg) with 200 ml water. Platelets were separated from plasma at baseline and 30 and 60 min after coffee drinking. Platelet aggregation was induced with three different agonists: collagen, arachidonic acid and ADP. Coffee drinking inhibited collagen (P < 0.05 from baseline at time 30 min) and arachidonic acid (P < 0.05 from baseline at time 60 min) induced platelet aggregation. Caffeine intake did not affect platelet aggregation induced by the three agonists. Coffee consumption induced a significant increase of platelet phenolic acids (likely present as glucuronate and sulphate derivatives), caffeic acid, the principal phenolic acid in coffee, raising from 0.3 (SEM 0.1) to 2.4 (SEM 0.6) ng/mg (P < 0.01). Caffeine was not detectable in platelets. Coffee drinking decreases platelet aggregation, and induces a significant increase in phenolic acid platelet concentration. The antiplatelet effect of coffee is independent from caffeine and could be a result of the interaction of coffee phenolic acids with the intracellular signalling network leading to platelet aggregation.

  14. pA2 values for antagonists of platelet activating factor on aggregation of rabbit platelets.

    PubMed Central

    O'Donnell, S. R.; Barnett, C. J.

    1988-01-01

    1. The relative potencies, and equilibrium dissociation constants, for nine antagonists of platelet activating factor (Paf) have been determined on rabbit platelets (in diluted platelet-rich plasma (PRP)) in experiments in which the aggregatory response to Paf was measured. 2. Log concentration-response (% maximum) curves to Paf were obtained in the absence (controls) and presence of different concentrations of each Paf antagonist drug. The antagonists shifted the Paf curves to a higher concentration range and the slopes of the Schild plots, constructed from these data, suggested that the drugs were competitive antagonists of Paf. The slopes of the Schild plots for CV-3988 and SRI 63-119 were greater than 1. 3. The pA2 values (pKB values in parentheses) were: WEB 2086 7.31 (7.63); SRI 63-119 6.95; L-652,731 6.71 (6.73); BN 52021 6.38 (6.47); SRI 63-072 6.36 (6.43); CV-3988 5.87; 48740 RP 4.97 (5.07); ketotifen 4.94 (4.95); thiazinamium 4.73 (4.76). 4. This study provides, for the first time, some functional response data for Paf antagonists (pKB values) which are in an appropriate form for use in classifying putative Paf receptors. The study also provides the comparative potencies of these Paf antagonists in inhibiting Paf-induced platelet aggregation. WEB 2086 was the most potent of the drugs examined. PMID:3293683

  15. Enhanced Shear-induced Platelet Aggregation Due to Low-temperature Storage

    DTIC Science & Technology

    2013-07-01

    rheometer. PLT aggregation was measured using a flow cytometer as an increase in the forward scatter-side scatter population which is bigger and...500, 2500, or 10,000/second for 120 seconds at 37°C, and PLT aggregation was measured. (A) Flow cytometric plots for the measurement of PLT...platelet aggregation and microaggregate formation in whole blood by flow cytometry. Platelets 2004;15:85-93. 26. Shankaran H, Alexandridis P

  16. Thrombotic thrombocytopenic purpura: from platelet aggregates to plasma.

    PubMed

    Marques, Marisa B; Mayfield, Charles A; Blackall, Douglas P

    2004-06-01

    Thrombotic thrombocytopenic purpura (TTP) is a syndrome of severe thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation. Although some patients also have a combination of fever and neurologic and/or renal manifestations, these are not required for the diagnosis. Thus, plasmapheresis should start as soon as TTP is placed high in the differential diagnosis to prevent significant mortality. Histopathologically, TTP is characterized by widespread platelet thrombi in the microcirculation. Ultralarge von Willebrand factor (vWf) multimers found in the patient's plasma are the basis for the platelet thrombi. Recent evidence has linked the abnormal fragments of vWf with deficiency of a plasma enzyme named vWf-cleaving protease, or ADAMTS-13. While a small percentage of patients with TTP have a constitutional defect in this enzyme, many with the acute idiopathic form have an antibody to ADAMTS-13, affecting its ability to cleave vWf. The determination of the enzyme activity and the presence of its inhibitor have emerged as a potential tool in the diagnosis and prognosis of TTP. Furthermore, it helps to differentiate TTP from the hemolytic uremic syndrome, in which the level of ADAMTS-13 is expected to be normal or only slightly decreased.

  17. Evidence for a Structural Requirement for the Aggregation of Platelets by Collagen

    PubMed Central

    Jaffe, Russell; Deykin, Daniel

    1974-01-01

    This study investigates whether soluble collagen can initiate platelet aggregation or whether a higher degree of polymerization is required. Purified rat skin collagen was prepared in four states. Soluble monomeric collagen, containing 2 μM calcium chloride, was maintained at 4°C until use. A previously uncharacterized form of collagen, soluble microfibrillar collagen, was prepared from monomeric collagen containing calcium chloride by allowing it to polymerize at 23°C. Viscometric and electron microscopic characterization of microfibrillar collagen indicated polymerization to ordered native filaments. Particulate native macrofibrillar collagen was prepared from monomeric collagen by allowing it to polymerize at 37°C in the absence of calcium. Particulate collagen, in which the fibers were randomly associated, was prepared by salt precipitation of calcium-free monomeric collagen. Microfibrillar and native macrofibrillar collagen initiated platelet aggregation, with a lag phase of approximately 60 s. Monomeric collagen initiated aggregation with a lag phase of approximately 180 s. The duration of the lag phase for platelet aggregation initiated by monomeric collagen was independent of the dose. Salt-precipitated particulate collagen did not initiate platelet aggregation. Agents which prolong the transition from monomeric collagen to fibrillar collagen (urea, arginine) retarded or prevented the aggregation of platelets by monomeric collagen. Sodium borohydride, which stabilizes the intraand intermolecular cross-links of collagen did not affect platelet aggregation. Penicillamine, which displaces the intermolecular cross-links and binds the intramolecular cross-links of collagen, did not prevent platelet aggregation. The data suggest that an architectural requirement exists for the initiation of self-perpetuating platelet aggregation; that tropocollagen units do not fulfill this requirement; that a soluble collagen preparation, microfibrillar collagen, contains

  18. Licochalcones extracted from Glycyrrhiza inflata inhibit platelet aggregation accompanied by inhibition of COX-1 activity

    PubMed Central

    Okuda-Tanino, Asa; Sugawara, Daiki; Tashiro, Takumi; Iwashita, Masaya; Obara, Yutaro; Moriya, Takahiro; Tsushima, Chisato; Saigusa, Daisuke; Tomioka, Yoshihisa; Ishii, Kuniaki; Nakahata, Norimichi

    2017-01-01

    Licochalcones extracted from Glycyrrhiza inflata are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A2 receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A2 induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 in vitro. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E2 synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity. PMID:28282426

  19. Licochalcones extracted from Glycyrrhiza inflata inhibit platelet aggregation accompanied by inhibition of COX-1 activity.

    PubMed

    Okuda-Tanino, Asa; Sugawara, Daiki; Tashiro, Takumi; Iwashita, Masaya; Obara, Yutaro; Moriya, Takahiro; Tsushima, Chisato; Saigusa, Daisuke; Tomioka, Yoshihisa; Ishii, Kuniaki; Nakahata, Norimichi

    2017-01-01

    Licochalcones extracted from Glycyrrhiza inflata are known to have a variety of biological properties such as anti-inflammatory, anti-bacterial, and anti-tumor activities, but their action on platelet aggregation has not yet been reported. Therefore, in this study we investigated the effects of licochalcones on platelet aggregation. Collagen and U46619, a thromboxane A2 receptor agonist, caused rabbit platelet aggregation, which was reversed by pretreatment with licochalcones A, C and D in concentration-dependent manners. Among these compounds, licochalcone A caused the most potent inhibitory effect on collagen-induced platelet aggregation. However, the licochalcones showed marginal inhibitory effects on thrombin or ADP-induced platelet aggregation. In addition to rabbit platelets, licochalcone A attenuated collagen-induced aggregation in human platelets. Because licochalcone A also inhibited arachidonic acid-induced platelet aggregation and production of thromboxane A2 induced by collagen in intact platelets, we further examined the direct interaction of licochalcone A with cyclooxygenase (COX)-1. As expected, licochalcone A caused an inhibitory effect on both COX-1 and COX-2 in vitro. Regarding the effect of licochalcone A on COX-1 enzyme reaction kinetics, although licochalcone A showed a stronger inhibition of prostaglandin E2 synthesis induced by lower concentrations of arachidonic acid, Vmax values in the presence or absence of licochalcone A were comparable, suggesting that it competes with arachidonic acid at the same binding site on COX-1. These results suggest that licochalcones inhibit collagen-induced platelet aggregation accompanied by inhibition of COX-1 activity.

  20. Effect of evening primrose oil on platelet aggregation in rabbits fed an atherogenic diet.

    PubMed

    De La Cruz, J P; Martín-Romero, M; Carmona, J A; Villalobos, M A; Sánchez de la Cuesta, F

    1997-07-01

    Evening primrose oil (Oenothera biennis) is a rich source of omega-6 series fatty acids. We report here the effects of dietary supplementation with evening primrose oil (EPO) on platelet aggregation as the main factor in arterial thrombus formation in an experimental model of atherogenesis in rabbits. A total of 40 male white New Zealand rabbits were divided into four groups (n = 10 animals/group): 1: normal diet, 2: atherogenic diet (ATD), 3: normal diet enriched with 15% EPO, 4: ATD + EPO. Each group was kept on the diet for 6 weeks. We determined serum lipid profile, platelet aggregation in whole blood, platelet thromboxane B2 production and platelet lipid peroxides. The atherogenic diet increased platelet aggregation (135% when ADP was used, and 185% when collagen was used as the inducer). Evening primrose oil reduced hyperaggregation to the values obtained in rabbits fed with the normal diet. Thromboxane synthesis was increased from 0.18 to 2.28 nmol/10(9) platelets); EPO reduced this value to 1.38 nmol/10(9) platelets. Lipid peroxides were increased by ATD from 0.27 to 0.81 nmol/10(8) platelets; EPO prevented this increase (0.35 nmol/10(8) platelets). In conclusion, EPO reduced platelet hyperaggregability in rabbits fed an atherogenic diet.

  1. Platelet Aggregation Unchanged by Lipoprotein-Associated Phospholipase A2 Inhibition: Results from an In Vitro Study and Two Randomized Phase I Trials

    PubMed Central

    Shaddinger, Bonnie C.; Xu, Yanmei; Roger, James H.; Macphee, Colin H.; Handel, Malcolm; Baidoo, Charlotte A.; Magee, Mindy; Lepore, John J.; Sprecher, Dennis L.

    2014-01-01

    Background We explored the theorized upregulation of platelet-activating factor (PAF)– mediated biologic responses following lipoprotein-associated phospholipase A2 (Lp-PLA2) inhibition using human platelet aggregation studies in an in vitro experiment and in 2 clinical trials. Methods and Results Full platelet aggregation concentration response curves were generated in vitro to several platelet agonists in human plasma samples pretreated with rilapladib (selective Lp-PLA2 inhibitor) or vehicle. This was followed by a randomized, double-blind crossover study in healthy adult men (n = 26) employing a single-agonist dose assay of platelet aggregation, after treatment of subjects with 250 mg oral rilapladib or placebo once daily for 14 days. This study was followed by a second randomized, double-blind parallel-group trial in healthy adult men (n = 58) also treated with 250 mg oral rilapladib or placebo once daily for 14 days using a full range of 10 collagen concentrations (0–10 µg/ml) for characterizing EC50 values for platelet aggregation for each subject. Both clinical studies were conducted at the GlaxoSmithKline Medicines Research Unit in the Prince of Wales Hospital, Sydney, Australia. EC50 values derived from multiple agonist concentrations were compared and no pro-aggregant signals were observed during exposure to rilapladib in any of these platelet studies, despite Lp-PLA2 inhibition exceeding 90%. An increase in collagen-mediated aggregation was observed 3 weeks post drug termination in the crossover study (15.4% vs baseline; 95% confidence interval [CI], 3.9–27.0), which was not observed during the treatment phase and was not observed in the parallel-group study employing a more robust EC50 examination. Conclusions Lp-PLA2 inhibition does not enhance platelet aggregation. Trial Registration 1) Study 1: ClinicalTrials.gov NCT01745458 2) Study 2: ClinicalTrials.gov NCT00387257 PMID:24475026

  2. Platelet concentrates transfusion in cardiac surgery in relation to preoperative point-of-care assessment of platelet adhesion and aggregation.

    PubMed

    Solomon, Cristina; Hartmann, Jennifer; Osthaus, Alexander; Schöchl, Herbert; Raymondos, Kostas; Koppert, Wolfgang; Rahe-Meyer, Niels

    2010-01-01

    Platelet dysfunction is an important cause of bleeding early after cardiac surgery. Whole-blood multiple electrode aggregometry (MEA), investigating the adhesion and aggregation of activated platelets onto metal electrodes, has shown correlations with platelet concentrates transfusion in this setting. Platelet activity in vivo is dependent on shear stress, an aspect that cannot be investigated with MEA, but with the cone and plate(let) analyzer (CPA) Impact-R that measures the interaction of platelets and von Willebrand factor (vWF) in whole blood under shear. We hypothesized that preoperative CPA may show better correlation with platelet concentrates transfusion post-cardiac surgery than MEA, since it is dependent on both platelet activity and platelet interaction with vWF multimers. Blood was obtained preoperatively from 30 patients undergoing aorto-coronary bypass (ACB) and 20 patients with aortic valve (AV) surgery. MEA was performed in hirudin-anticoagulated blood. The Impact-R analyses were performed in blood anticoagulated with hirudin, heparin or the standard anticoagulant citrate. For the light microscopy images obtained, the parameter surface coverage (SC) was calculated. Preoperative Impact-R results were abnormally decreased in AV patients and significantly lower than in ACB patients. For the Impact-R analysis performed in citrated blood, no correlation with platelet concentrates transfusion was observed. In contrast, MEA was comparable between the groups and correlated significantly with intraoperative platelet concentrates transfusion in both groups (rho between -0.47 and -0.62, p < 0.05). Multiple electrode aggregometry appeared more useful and easier to apply than CPA for preoperatively identifying patients with platelet concentrates transfusion in cardiac surgery.

  3. In vitro platelet activation, aggregation and platelet-granulocyte complex formation induced by surface modified single-walled carbon nanotubes.

    PubMed

    Fent, János; Bihari, Péter; Vippola, Minnamari; Sarlin, Essi; Lakatos, Susan

    2015-08-01

    Surface modification of single-walled carbon nanotubes (SWCNTs) such as carboxylation, amidation, hydroxylation and pegylation is used to reduce the nanotube toxicity and render them more suitable for biomedical applications than their pristine counterparts. Toxicity can be manifested in platelet activation as it has been shown for SWCNTs. However, the effect of various surface modifications on the platelet activating potential of SWCNTs has not been tested yet. In vitro platelet activation (CD62P) as well as the platelet-granulocyte complex formation (CD15/CD41 double positivity) in human whole blood were measured by flow cytometry in the presence of 0.1mg/ml of pristine or various surface modified SWCNTs. The effect of various SWCNTs was tested by whole blood impedance aggregometry, too. All tested SWCNTs but the hydroxylated ones activate platelets and promote platelet-granulocyte complex formation in vitro. Carboxylated, pegylated and pristine SWCNTs induce whole blood aggregation as well. Although pegylation is preferred from biomedical point of view, among the samples tested by us pegylated SWCNTs induced far the most prominent activation and a well detectable aggregation of platelets in whole blood.

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

    PubMed

    Navdaev, Alexey; Subramanian, Hariharan; Petunin, Alexey; Clemetson, Kenneth J; Gambaryan, Stepan; Walter, Ulrich

    2014-01-01

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

  5. Surface morphology of platelet adhesion influenced by activators, inhibitors and shear stress

    NASA Astrophysics Data System (ADS)

    Watson, Melanie Groan

    Platelet activation involves multiple events, one of which is the generation and release of nitric oxide (NO), a platelet aggregation inhibitor. Platelets simultaneously send and receive various agents that promote a positive and negative feedback control system during hemostasis. Although the purpose of platelet-derived NO is not fully understood, NO is known to inhibit platelet recruitment. NO's relatively large diffusion coefficient allows it to diffuse more rapidly than platelet agonists. It may thus be able to inhibit recruitment of platelets near the periphery of a growing thrombus before agonists have substantially accumulated in those regions. Results from two studies in our laboratory differed in the extent to which platelet-derived NO decreased platelet adhesion. Frilot studied the effect of L-arginine (L-A) and NG-Methyl-L-arginine acetate salt (L-NMMA) on platelet adhesion to collagen under static conditions in a Petri dish. Eshaq examined the percent coverage on collagen-coated and fibrinogen-coated microchannels under shear conditions with different levels of L-A and Adenosine Diphosphate (ADP). Frilot's results showed no effect of either L-A or L-NMMA on surface coverage, thrombus size or serotonin release, while Eshaq's results showed a decrease in surface coverage with increased levels of L-A. A possible explanation for these contrasting results is that platelet-derived NO may be more important under flow conditions than under static conditions. For this project, the effects of L-A. ADP and L-NMMA on platelet adhesion were studied at varying shear stresses on protein-coated glass slides. The surface exposed to platelet-rich-plasma in combination with each chemical solution was observed under AFM, FE-SEM and fluorescence microscopy. Quantitative and qualitative comparisons of images obtained with these techniques confirmed the presence of platelets on the protein coatings. AFM images of fibrinogen and collagen-coated slides presented characteristic

  6. Cholesterol-induced stimulation of platelet aggregation is prevented by a hempseed-enriched diet.

    PubMed

    Prociuk, M A; Edel, A L; Richard, M N; Gavel, N T; Ander, B P; Dupasquier, C M C; Pierce, G N

    2008-04-01

    Hypercholesterolemia indirectly increases the risk for myocardial infarction by enhancing the ability of platelets to aggregate. Diets enriched with polyunsaturated fatty acids (PUFAs) have been shown to reduce the detrimental effects of cholesterol on platelet aggregation. This study investigated whether dietary hempseed, a rich source of PUFAs, inhibits platelet aggregation under normal and hypercholesterolemic conditions. Male New Zealand white rabbits were fed one of 6 dietary interventions: regular control diet (RG); control diet + 10% hempseed (HP); control diet + 10% partially delipidated hempseed (DHP); control diet + 0.5% cholesterol (OL); control diet + 0.5% cholesterol + 10% hempseed (OLHP); control diet + 5% coconut oil (CO). After 8 weeks, blood was collected to measure ADP- and collagen-induced platelet aggregation and plasma levels of fatty acids, cholesterol, and triglycerides. The hempseed-fed animals (HP and OLHP) displayed elevated plasma levels of PUFAs and a prominent enhancement in 18:3n-6 (gamma-linolenic acid, GLA) levels, a unique PUFA found in hempseed. The cholesterol-supplemented groups (OL and OLHP) had significantly elevated plasma levels of cholesterol and triglycerides, but platelet aggregation was significantly augmented only in the OL group. The addition of hempseed to this diet (OLHP) normalized aggregation. The direct addition of GLA to the OL platelet samples blocked the cholesterol-induced stimulation of platelet aggregation. The results of this study demonstrate that when hempseed is added to a cholesterol-enriched diet, cholesterol-induced platelet aggregation returns to control levels. This normalization is not due to a reduction in plasma cholesterol levels, but may be partly due to increased levels of plasma GLA.

  7. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress.

    PubMed Central

    Ikeda, Y; Handa, M; Kawano, K; Kamata, T; Murata, M; Araki, Y; Anbo, H; Kawai, Y; Watanabe, K; Itagaki, I

    1991-01-01

    Exposure of platelets to shear stress leads to aggregation in the absence of exogenous agonists. We have now found that different adhesive proteins and platelet membrane glycoproteins are involved in aggregation depending on the shear stress conditions and the concentration of divalent cations in the medium. When blood is collected with trisodium citrate as anticoagulant, which causes a decrease in the levels of external ionized calcium ([Ca2+]o), platelet aggregation can be induced under low shear force (12 dyn/cm2) and is mediated by fibrinogen binding to the glycoprotein IIb-IIIa complex. Aggregates formed under these conditions are not stable, and when shear force is increased to 68 dyn/cm2, disaggregation results. By contrast, platelets from blood collected with hirudin as anticoagulant, wherein [Ca2+]o is within normal plasma levels, do not undergo low shear-induced aggregation; however, after exposure to a shear force above 80 dyn/cm2, aggregation is observed but only when von Willebrand factor is present and can interact with both its platelet binding sites, glycoprotein Ib-IX and glycoprotein IIb-IIIa. Fibrinogen is not involved in high shear-induced aggregation which, in fact, occurs normally in patients with severe afibrinogenemia. Thus, von Willebrand factor in the absence of exogenous agonists can mediate platelet aggregation in experimental conditions that may mimic the hemorheological situation of partially occluded arteries. This pathway of platelet aggregation involving only one adhesive ligand and two membrane adhesion receptors may play a relevant role in thrombogenesis. PMID:2010539

  8. Duration of exposure to high fluid shear stress is critical in shear-induced platelet activation-aggregation.

    PubMed

    Zhang, Jian-ning; Bergeron, Angela L; Yu, Qinghua; Sun, Carol; McBride, Latresha; Bray, Paul F; Dong, Jing-fei

    2003-10-01

    Platelet functions are increasingly measured under flow conditions to account for blood hydrodynamic effects. Typically, these studies involve exposing platelets to high shear stress for periods significantly longer than would occur in vivo. In the current study, we demonstrate that the platelet response to high shear depends on the duration of shear exposure. In response to a 100 dyn/cm2 shear stress for periods less than 10-20 sec, platelets in PRP or washed platelets were aggregated, but minimally activated as demonstrated by P-selectin expression and binding of the activation-dependent alphaIIbbeta3 antibody PAC-1 to sheared platelets. Furthermore, platelet aggregation under such short pulses of high shear was subjected to rapid disaggregation. The disaggregated platelets could be re-aggregated by ADP in a pattern similar to unsheared platelets. In comparison, platelets that are exposed to high shear for longer than 20 sec are activated and aggregated irreversibly. In contrast, platelet activation and aggregation were significantly greater in whole blood with significantly less disaggregation. The enhancement is likely via increased collision frequency of platelet-platelet interaction and duration of platelet-platelet association due to high cell density. It may also be attributed to the ADP release from other cells such as red blood cells because increased platelet aggregation in whole blood was partially inhibited by ADP blockage. These studies demonstrate that platelets have a higher threshold for shear stress than previously believed. In a pathologically relevant timeframe, high shear alone is likely to be insufficient in inducing platelet activation and aggregation, but acts synergistically with other stimuli.

  9. 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.

  10. Rapidly regulating platelet activity in vivo with an antidote controlled platelet inhibitor.

    PubMed

    Nimjee, Shahid M; Lohrmann, Jens D; Wang, Haichen; Snyder, David J; Cummings, Thomas J; Becker, Richard C; Oney, Sabah; Sullenger, Bruce A

    2012-02-01

    Millions of individuals are prescribed platelet inhibitors, such as aspirin and clopidogrel, to reduce their risk of thrombosis-related clinical events. Unfortunately many platelet inhibitors are contraindicated in surgical settings because of their inherent bleeding risk complicating the treatment of patients who require surgery. We describe the development of a potent antiplatelet agent, an RNA aptamer-termed Ch-9.14-T10 that binds von Willebrand factor (VWF) with high affinity and inhibits thrombosis in a murine carotid artery damage model. As expected, when this potent antiplatelet agent is administered, it greatly increases bleeding from animals that are surgically challenged. To improve this antiplatelet agent's safety profile, we describe the generation of antidotes that can rapidly reverse the activity of Ch-9.14-T10 and limit blood loss from surgically challenged animals. Our work represents the first antidote controllable antiplatelet agent, which could conceivably lead to improved medical management of patients requiring antiplatelet medication who also need surgery.

  11. Perilla oil improves blood flow through inhibition of platelet aggregation and thrombus formation

    PubMed Central

    Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang-Sei; Lee, Sung-Pyo; Kang, Myung-Hwa; Choi, Ehn-Kyoung

    2014-01-01

    The inhibitory effects of perilla oil on the platelet aggregation in vitro and thrombosis in vivo were investigated in comparison with aspirin, a well-known blood flow enhancer. Rabbit platelet-rich plasma was incubated with perilla oil and aggregation inducers collagen or thrombin, and the platelet aggregation rate was analyzed. Perilla oil significantly inhibited both the collagen- and thrombin-induced platelet aggregations, in which the thromboxane B2 formation from collagen-activated platelets were reduced in a concentration-dependent manner. Rats were administered once daily by gavage with perilla oil for 1 week, carotid arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Perilla oil delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 0.5 mL/kg. In addition, a high dose (2 mL/kg) of perilla oil greatly prevented the occlusion, comparable to the effect of aspirin (30 mg/kg). The results indicate that perilla oil inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is proposed that perilla oil could be a good candidate without adverse effects for the improvement of blood flow. PMID:24707301

  12. Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation

    PubMed Central

    Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung

    2013-01-01

    The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow. PMID:24396387

  13. Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation.

    PubMed

    Jang, Ja-Young; Kim, Tae-Su; Cai, Jingmei; Kim, Jihyun; Kim, Youngeun; Shin, Kyungha; Kim, Kwang Sei; Park, Sung Kyeong; Lee, Sung-Pyo; Choi, Ehn-Kyoung; Rhee, Man Hee; Kim, Yun-Bae

    2013-12-01

    The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

  14. Inhibition of the activation of Hageman factor (factor XII) and of platelet aggregation by extracts of Brugia malayi microfilariae.

    PubMed

    Foster, C B; Flanigan, T P; Kazura, J W; Dumenco, L L; Ratnoff, O D

    1991-05-01

    In human filariasis, large numbers of blood-borne microfilariae circulate unimpeded through the blood stream. How intravascular filarial parasites avoid precipitating thrombosis has not been studied in detail. We hypothesized that extracts of Brugia malayi microfilariae would contain factors that inhibit activation of hemostatic mechanisms. Initial studies demonstrated an inhibitor specific for the intrinsic coagulation cascade. The addition of microfilarial extracts to human plasma prolonged the activated partial thromboplastin time in a dose-dependent fashion but did not prolong the prothrombin, thrombin, or Russell's viper venom times. Microfilarial extracts (0.1 mg/ml) completely inhibited activation of Hageman factor (factor XII, at 0.05 U/ml) as measured in an amidolytic assay. Hageman factor previously activated by ellagic acid (factor XIIa) retained full enzymatic activity in the presence of microfilarial extract (0.1 mg/ml). The presence of inhibitory activity in the culture medium of live parasites raises the possibility that microfilariae secrete an inhibitory protein into their local environment. Microfilarial extracts at a final concentration of 0.1 mg/ml also inhibited collagen- and adenosine diphosphate-induced platelet aggregation. Arachidonic acid-induced platelet aggregation was inhibited by microfilarial extracts at a final concentration of 0.6 mg/ml. These results suggest that microfilariae of Brugia malayi, a human filarial parasite, may avoid initiating thrombosis through inhibition of the intrinsic coagulation pathway and platelet aggregation.

  15. Activation of Protein Kinase G (PKG) Reduces Neointimal Hyperplasia, Inhibits Platelet Aggregation, and Facilitates Re-endothelialization

    PubMed Central

    Kim, Ju-Young; Yang, Han-Mo; Lee, Joo-Eun; Kim, Baek-Kyung; Jin, Sooryeonhwa; Lee, Jaewon; Park, Kyung-Woo; Cho, Hyun-Jai; Kwon, Yoo-Wook; Lee, Hae-Young; Kang, Hyun-Jae; Oh, Byung-Hee; Park, Young-Bae; Kim, Hyo-Soo

    2016-01-01

    In spite of its great success in reducing restenosis, drug-eluting stent (DES) has unfavorable aspects such as stent thrombosis and delayed re-endothelialization. We examined the effects of PKG activation by Exisulind on neointimal formation, platelet aggregation, and re-endothelialization. Exisulind significantly reduced VSMCs viability, cell cycle progression, migration, and neointimal hyperplasia after vascular injury in rat carotid arteries. Interestingly, in contrast to the effect on VSMC viability, Exisulind did not reduce the viability of endothelial cells. Increased PKG activity by Exisulind inhibited PDGF-stimulated phenotype change of VSMCs from a contractile to a synthetic form. Conversely, the use of PKG inhibitor or gene transfer of dominant-negative PKG reversed the effects of Exisulind, resulting in the increased viability of VSMCs and neointimal formation. In addition, Exisulind facilitated the differentiation of peripheral blood mononuclear cells to endothelial lineage via PKG pathway, while inhibiting to VSMCs lineage, which was correlated with the enhanced re-endothelialization in vivo. Finally, Exisulind reduced platelet aggregation, which was mediated via PKG activation. This study demonstrated that Exisulind inhibits neointimal formation and platelet aggregation while increasing re-endothelialization via PKG pathway. These findings suggest that Exisulind could be a promising candidate drug of DES for the prevention of restenosis without other complications. PMID:27833146

  16. Hydrodynamic effects and receptor interactions of platelets and their aggregates in linear shear flow.

    PubMed Central

    Tandon, P; Diamond, S L

    1997-01-01

    We have modeled platelet aggregation in a linear shear flow by accounting for two body collision hydrodynamics, platelet activation and receptor biology. Considering platelets and their aggregates as unequal-sized spheres with DLVO interactions (psi(platelet) = -15 mV, Hamaker constant = 10(-19) J), detailed hydrodynamics provided the flow field around the colliding platelets. Trajectory calculations were performed to obtain the far upstream cross-sectional area and the particle flux through this area provided the collision frequency. Only a fraction of platelets brought together by a shearing fluid flow were held together if successfully bound by fibrinogen cross-bridging GPIIb/IIIa receptors on the platelet surfaces. This fraction was calculated by modeling receptor-mediated aggregation using the formalism of Bell (Bell, G. I. 1979. A theoretical model for adhesion between cells mediated by multivalent ligands. Cell Biophys. 1:133-147) where the forward rate of bond formation dictated aggregation during collision and was estimated from the diffusional limited rate of lateral association of receptors multiplied by an effectiveness factor, eta, to give an apparent rate. For a value of eta = 0.0178, we calculated the overall efficiency (including both receptor binding and hydrodynamics effects) for equal-sized platelets with 50,000 receptors/platelet to be 0.206 for G = 41.9 s(-1), 0.05 for G = 335 s(-1), and 0.0086 for G = 1920 s(-1), values which are in agreement with efficiencies determined from initial platelet singlet consumption rates in flow through a tube. From our analysis, we predict that bond formation proceeds at a rate of approximately 0.1925 bonds/microm2 per ms, which is approximately 50-fold slower than the diffusion limited rate of association. This value of eta is also consistent with a colloidal stability of unactivated platelets at low shear rates. Fibrinogen was calculated to mediate aggregation quite efficiently at low shear rates but not at

  17. Influence of combinations of acetylsalicylic acid, acetaminophen, and diclofenac on platelet aggregation.

    PubMed

    Galliard-Grigioni, Katja S; Fehr, Martin; Reinhart, Walter H

    2008-10-24

    Acetylsalicylic acid (aspirin) is often given together with other nonsteroidal anti-inflammatory drugs and acetaminophen. The latter have been accused in epidemiologic studies to cause an increased cardiovascular risk. We have, therefore, analysed the influence of various such drug combinations on platelet aggregation in vitro. Citrated blood was incubated with either 25 microg/ml acetaminophen, 0.5 microg/ml aspirin, 0.04 microg/ml diclofenac, or buffer; followed by a second of the above-mentioned solutions. After a 20 min incubation, platelet aggregation was assessed with a platelet function analyser (PFA-100), which measures the pore closure time (CT) by aggregating platelets. The length of CT reflects the degree of platelet inhibition. Acetaminophen alone did not affect platelet aggregation. Aspirin and diclofenac both increased CT (184+/-69 s, P<0.01 and 196+/-54 s, P<0.001; control 120+/-13 s). Combinations of either aspirin and diclofenac, aspirin and acetaminophen, or diclofenac and acetaminophen increased CT further (290+/-22 s, 281+/-36 s, 288+/-25 s, respectively, P<0.001). The time sequence of drug application was important: when diclofenac or acetaminophen was added before aspirin, platelet aggregation was less inhibited than when given in opposite order, i.e. aspirin prior to diclofenac or acetaminophen. We conclude that acetaminophen by itself does not affect platelet aggregation, but potentiates the antiaggregatory effect of aspirin or diclofenac. Aspirin given before acetaminophen or diclofenac had a more potent antiaggregatory effect than vice versa. These observations may have clinical implications.

  18. Paraphenylene diamine exacerbates platelet aggregation and thrombus formation in response to a low dose of collagen.

    PubMed

    Zaid, Younes; Marhoume, Fatimazahra; Senhaji, Nezha; Kojok, Kevin; Boufous, Hicham; Naya, Abdallah; Oudghiri, Mounia; Darif, Youssef; Habti, Norddine; Zouine, Soukaina; Mohamed, Fekhaoui; Chait, Abderahmane; Bagri, Abdellah

    2016-02-01

    Paraphenylene daimine (PPD) is an aromatic amine that is widely used in several industrial products; however, its toxicity has been reported in several cases of cardiac arrests. As platelets play a key role in cardiovascular diseases, we aimed to determine the impact of PPD in vitro and in vivo on platelet function. Our findings demonstrated that platelet activation and aggregation were strongly enhanced by PPD. Treatment with PPD primed human platelets that became more reactive in response to low doses of collagen. Furthermore, PPD exacerbated thrombus formation in rats in comparison with those untreated. Our results suggest that PPD is an important platelet primer predisposing platelets to promote thrombus formation in response to vascular injury. This should prompt the authorities to consider controlling the marketing of this product.

  19. The passage of a hemodialysis filter affects hemorheology, red cell shape, and platelet aggregation.

    PubMed

    Reinhart, W H; Cagienard, F; Schulzki, T; Venzin, R M

    2014-01-01

    We investigated the influence of the passage of a hemodialysis filter on red blood cells (RBCs), platelets, and hemorheological parameters. After one hour of hemodialysis, blood was drawn from 15 patients immediately ahead and behind the dialysis filter. RBCs were fixed for morphological analysis. Blood viscosity was measured with a Couette viscometer (LS-30, Contraves), RBC aggregation with a Myrenne aggregometer, platelet aggregation in flowing whole blood and in platelet rich plasma. The passage of the hemodialysis filter increased the hematocrit from 34.0 ± 3.8 to 44.6 ± 8.7% (p < 0.01). Discocytes decreased from 73 ± 9 to 60 ± 15%, while echinocytes/knizocytes were more abundant 24 ± 9% and 38 ± 15%, respectively, p < 0.01). Blood viscosity increased from 3.77 ± 0.52 to 6.75 ± 2.21 mPa.s (p < 0.01). The RBC aggregation index decreased from 25.8 ± 5.0 to 20.9 ± 5.6 (p < 0.05). These changes were less pronounced when the blood flow rate was reduced from 350 to 100 ml/min. Platelet aggregation was slightly increased in flowing whole blood, but decreased in platelet rich plasma. At the end of hemodialysis, a small increase in abnormally shaped RBCs, hematocrit, and whole blood viscosity persisted; platelet aggregation in flowing whole blood was reduced in all patients. We conclude that the passage of a hemodialysis filter induced RBC shape changes, increased the hematocrit, whole blood and plasma viscosity, decreased RBC aggregation, and affected platelet aggregation.

  20. Comparison of different procedures to prepare platelet-rich plasma for studies of platelet aggregation by light transmission aggregometry.

    PubMed

    Femia, Eti Alessandra; Pugliano, Mariateresa; Podda, Gianmarco; Cattaneo, Marco

    2012-01-01

    Light transmission aggregometry (LTA), the gold standard for the study of patients with defects of platelet function, is a poorly standardized technique. The guidelines that have been produced so far are largely based on consensus of experts, due to the absence of studies directly comparing different procedures. Therefore, ad hoc studies are needed to gather scientific evidence on how to choose the most appropriate procedures for LTA measurement. In this study, we aimed at evaluating the most appropriate conditions for preparing samples of platelet-rich plasma (PRP) for studies of platelet aggregation by LTA. Citrate-anticoagulated blood from 32 individuals was centrifuged at 150, 200, 250 or 300×g at room temperature for 10 min. Red blood cells contamination was highest in PRP prepared at 150×g; mean platelet volume (MPV) was lowest in PRP prepared at 300×g. The extent of platelet aggregation measured by LTA was lower and more variable in PRP prepared at 300×g. Therefore, centrifugation of blood at 200×g or 250×g for 10 min appears to be the best condition for preparing PRP for LTA studies.

  1. 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

  2. Platelet-aggregating activity of released factor(s) from Trypanosoma brucei brucei.

    PubMed

    Nwagwu, M; Inyang, A L; Molokwu, R I; Essien, E M

    1989-12-01

    The effect of factors derived from Trypanosoma brucei brucei on rat platelets was studied. T. brucei at a concentration of 4 X 10(9) trypanosomes/ml phosphate saline glucose (PSG) was stored at -20 degrees C for 18 h, thawed, and a supernatant fraction, trypanosome-derived supernatant (TDS) was obtained by spinning the sample at 3000 g for 10 min at 20 degrees C. Normal rat platelets, prepared as platelet-rich plasma (PRP), were then incubated with TDS in the absence or presence of ADP (0.05-0.1 microM). The results showed that approximately 83% platelet aggregation was induced by addition of TDS (50 microliters; 113 micrograms protein) to 100 microliters PRP with a platelet count of 10(6). simultaneous addition of ADP and TDS to PRP produced a synergistic effect. It was also shown that a supernatant fraction, obtained by incubating live T. brucei (4 X 10(9)/microliters PSG) at 0 degrees C 1 h and spinning down the trypanosomes (3000 g for 10 min), also induced platelet aggregation. The nature of the factor(s) derived from, or released by, T. brucei inducing platelet aggregation is being investigated but it has been shown not to be ADP.

  3. Platelet calcium and quenched-flow aggregation kinetics in essential hypertension.

    PubMed

    Taylor, M A; Ayers, C R; Gear, A R

    1989-06-01

    Abnormal platelet function may contribute to the complications of essential hypertension. We have studied the kinetics of platelet aggregation induced by adenosine diphosphate (ADP) or epinephrine, plasma beta-thromboglobulin, and basal, cytosolic, and free calcium, as correlates of platelet function. Fifteen untreated patients with essential hypertension and without detectable atherosclerosis, 18-40 years old, were compared with 30 matched normotensive control subjects. Maximal rates of platelet aggregation (Vmax) with ADP and epinephrine were significantly higher in patients than in control subjects (p less than 0.03), as assessed by quenched-flow aggregometry. However, significance was lost when Vmax was corrected for the platelet count. Paradoxically, the activation constants (Ka) for ADP were higher in patients than in control subjects (p less than 0.03). With ADP as the inducing agent, onset time (t) or lag period before aggregation begins was longer in patients than in control subjects (p less than 0.02). beta-thromboglobulin levels, an index of in vivo platelet activation, were not significantly different between the two groups (p = 0.13). The mean platelet cytosolic free calcium concentration was higher in patients (213 +/- 19 nM) than in control subjects (172 +/- 14 nM), but this difference was not statistically significant (p = 0.07). However, there was a close correlation between the free calcium level and systolic, diastolic, and mean blood pressure (p less than 0.003, p less than 0.04, p less than 0.004, respectively). No difference in platelet volume between the two groups was found. Our data suggest that platelets in the early stages of essential hypertension display an overall increased aggregation potential but a diminished sensitivity to ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Platelet Aggregation and Mental Stress Induced Myocardial Ischemia: Results from the REMIT Study

    PubMed Central

    Jiang, Wei; Boyle, Stephen H.; Ortel, Thomas L.; Samad, Zainab; Velazquez, Eric J.; Harrison, Robert W.; Wilson, Jennifer; Kuhn, Cynthia; Williams, Redford B.; O’Connor, Christopher M.; Becker, Richard C.

    2015-01-01

    BACKGROUND Mental stress-induced myocardial ischemia (MSIMI) is common in patients with ischemic heart disease (IHD) and associated with a poorer cardiovascular prognosis. Platelet hyperactivity is an important factor in acute coronary syndrome. This study examined associations between MSIMI and resting and mental stress-induced platelet activity. METHODS Eligible patients with clinically stable IHD underwent a battery of 3 mental stress tests during the recruitment phase of REMIT (Responses of Myocardial Ischemia to Escitalopram Treatment) study. MSIMI was assessed by echocardiography and electrocardiography. Ex vivo platelet aggregation in response to ADP, epinephrine, collagen, serotonin, and combinations of serotonin plus ADP, epinephrine, and collagen were evaluated as was platelet serotonin transporter expression. RESULTS Of the 270 participants who completed mental stress testing, and had both resting and post-stress platelet aggregation evaluation, 43.33% (N=117) met criteria for MSIMI and 18.15% (N=49) had normal left ventricular response to stress (NLVR). The MSIMI group, relative to the NLVR groups, demonstrated heightened mental stress-induced aggregation responses, as measured by area under the curve, to collagen 10 μM (6.95[5.54] vs. −14.23[8.75].; p=0.045), epinephrine 10 μM (12.84[4.84] vs. −6.40[7.61].; p=0.037) and to serotonin 10 μM plus ADP 1 μM (6.64[5.29] vs. −27.34[8.34]; p < .001). The resting platelet aggregation and serotonin transporter expression, however, were not different between the two groups. CONCLUSIONS These findings suggest that the dynamic change of platelet aggregation caused by mental stress may underlie MSIMI. While the importance of these findings requires additional investigation, they raise concern given the recognized relationship between mental stress-induced platelet hyperactivity and cardiovascular events in patients with IHD. PMID:25819856

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

    SciTech Connect

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

    1990-07-01

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

  6. Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability*

    PubMed Central

    Golebiewska, Ewelina M.; Harper, Matthew T.; Williams, Christopher M.; Savage, Joshua S.; Goggs, Robert; Fischer von Mollard, Gabriele; Poole, Alastair W.

    2015-01-01

    Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8−/− platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8−/− mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis. PMID:25404741

  7. Evaluation of Anti-Platelet Aggregation Effect of Some Allium Species

    PubMed Central

    Lorigooini, Zahra; Ayatollahi, Seyed Abdolmajid; Amidi, Salimeh; Kobarfard, Farzad

    2015-01-01

    Epidemiologic studies show that the cardiovascular diseases are associated with multiple factors such as raised serum total cholesterol, increased LDL, increased platelet aggregation, hypertension and smoking. In-vitro studies have confirmed the ability of some plants of Allium species to reduce these parameters. Therefore, we evaluated anti-platelet aggregation effect of some Allium species (Allium ampeloprasum, A. hirtifolium, A. haemanthoides, A. vavillovi, A. atroviolaceum, A. jesdianum, A. shelkovnikovii) using arachidonic acid (AA) and adenosine diphosphate (ADP) as platelet aggregation inducers. The screening results for methanolic extract of Allium species showed that the maximum effect of anti-platelet aggregation was related to A. atroviolaceum. This extract inhibited the in-vitro platelet aggregation induced by AA and ADP with IC50 values of 0.4881 (0.4826-0.4937) mg/ml and 0.4945 (0.4137-0.5911) mg/ml respectively. These results support the hypothesis that the dietary intake of Allium could be beneficial for prevention of cardiovascular diseases. PMID:26664390

  8. Platelet aggregation and serum adenosine deaminase (ADA) activity in pregnancy associated with diabetes, hypertension and HIV.

    PubMed

    Leal, Claudio A M; Leal, Daniela B R; Adefegha, Stephen A; Morsch, Vera M; da Silva, José E P; Rezer, João F P; Schrekker, Clarissa M L; Abdalla, Faida H; Schetinger, Maria R C

    2016-07-01

    Platelet aggregation and adenosine deaminase (ADA) activity were evaluated in pregnant women living with some disease conditions including hypertension, diabetes mellitus and human immunodeficiency virus infection. The subject population is consisted of 15 non-pregnant healthy women [control group (CG)], 15 women with normal pregnancy (NP), 7 women with hypertensive pregnancy (HP), 10 women with gestational diabetes mellitus (GDM) and 12 women with human immunodeficiency virus-infected pregnancy (HIP) groups. The aggregation of platelets was checked using an optical aggregometer, and serum ADA activity was determined using the colorimetric method. After the addition of 5 µM of agonist adenosine diphosphate, the percentage of platelet aggregation was significantly (p < 0·05) increased in NP, HP, GDM and HIP groups when compared with the CG, while the addition of 10 µM of the same agonist caused significant (p < 0·05) elevations in HP, GDM and HIP groups when compared with CG. Furthermore, ADA activity was significantly (p < 0·05) enhanced in NP, HP, GDM and HIP groups when compared with CG. In this study, the increased platelet aggregation and ADA activity in pregnancy and pregnancy-associated diseases suggest that platelet aggregation and ADA activity could serve as peripheral markers for the development of effective therapy in the maintenance of homeostasis and some inflammatory process in these pathophysiological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Development of tau aggregation inhibitors for Alzheimer's disease.

    PubMed

    Bulic, Bruno; Pickhardt, Marcus; Schmidt, Boris; Mandelkow, Eva-Maria; Waldmann, Herbert; Mandelkow, Eckhard

    2009-01-01

    A variety of human diseases are suspected to be directly linked to protein misfolding. Highly organized protein aggregates, called amyloid fibrils, and aggregation intermediates are observed; these are considered to be mediators of cellular toxicity and thus attract a great deal of attention from investigators. Neurodegenerative pathologies such as Alzheimer's disease account for a major part of these protein misfolding diseases. The last decade has witnessed a renaissance of interest in inhibitors of tau aggregation as potential disease-modifying drugs for Alzheimer's disease and other "tauopathies". The recent report of a phase II clinical trial with the tau aggregation inhibitor MTC could hold promise for the validation of the concept. This Review summarizes the available data concerning small-molecule inhibitors of tau aggregation from a medicinal chemistry point of view.

  10. Mathematical model and numerical method for studying platelet adhesion and aggregation during blood clotting

    SciTech Connect

    Fogelson, A.L.

    1984-10-01

    The repair of small blood vessels and the pathological growth of internal blood clots involve the formation of platelet aggregates adhering to portions of the vessel wall. Our microscopic model represents blood by a suspension of discrete massless platelets in a viscous incompressible fluid. Platelets are initially noncohesive; however, if stimulated by an above-threshold concentration of the chemical ADP or by contact with the adhesive injured region of the vessel wall, they become cohesive and secrete more ADP into the fluid. Cohesion between platelets and adhesion of a platelet to the injured wall are modeled by creating elastic links. Repulsive forces prevent a platelet from coming too close to another platelet or to the wall. The forces affect the fluid motion in the neighborhood of an aggregate. The platelets and secreted ADP both move by fluid advection and diffusion. The equations of the model are studied numerically in two dimensions. The platelet forces are calculated implicitly by minimizing a nonlinear energy function. Our minimization scheme merges Gill and Murray's (Math. Programming 7 (1974), 311) modified Newton's method with elements of the Yale sparse matix package. The stream-function formulation of the Stokes' equations for the fluid motion under the influence of platelet forces is solved using Bjorstad's biharmonic solver (''Numerical Solution of the Biharmonic Equation,'' Ph.D. Thesis, Stanford University, 1980). The ADP transport equation is solved with an alternating-direction implicit scheme. A linked-list data structure is introduced to keep track of changing platelet states and changing configurations of interplatelet links.

  11. Long-term mTOR inhibitors administration evokes altered calcium homeostasis and platelet dysfunction in kidney transplant patients

    PubMed Central

    López, Esther; Berna-Erro, Alejandro; Bermejo, Nuria; Brull, José María; Martinez, Rocío; Garcia Pino, Guadalupe; Alvarado, Raul; Salido, Ginés María; Rosado, Juan Antonio; Cubero, Juan José; Redondo, Pedro Cosme

    2013-01-01

    The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti-calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long-term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura-2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time-dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long-term administration of rapamycin to kidney transplant patients evokes alteration in platelet function. PMID:23577651

  12. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  13. Inhibition of Platelet Aggregation by Supernates from Stored Red Blood Cells

    DTIC Science & Technology

    2010-04-01

    platelet aggregates in fresh whole blood.[10] In those experiments , we observed that platelets in blood incubated with supernates from stored RBC...volumes in sterile cryovials, and the vials were stored at -80C. For each experiment , aliquots were thawed quickly in a 37°C water bath just before...The final ratio of whole blood to supernate was 2:1. For each experiment , blood was collected first into one red top Vacutainer tube (no anti

  14. Inhibitory effects of yuzu and its components on human platelet aggregation.

    PubMed

    Kim, Tae-Ho; Kim, Hye-Min; Park, Se Won; Jung, Yi-Sook

    2015-03-01

    Our previous study demonstrated that yuzu has an anti-platelet effect in rat blood. In the present study, we examined whether the anti-platelet effect of yuzu can be extended to human blood by investigating its ability to inhibit aggregations induced by various agonists in human platelet rich plasma (PRP). This study also investigated the underlying mechanism of yuzu focusing on ADP granule secretion, TXB2 formations, and PLCγ/Akt signaling. The results from this study showed that ethanolic yuzu extract (YE), and its components, hesperidin and naringin, inhibited human platelet aggregation in a concentration-dependent manner. YE, hesperidin and naringin also inhibited TXB2 formation and ADP release. The phosphorylation of PLCγ and Akt was significantly inhibited by YE, heperidin and naringin. Furthermore, we demonstrated that YE, heperidin and naringin has anti-platelet effects in rat ex vivo studies, and lower side effects in mice tail bleeding time studies. The results from this study suggest that YE, hesperidin and naringin can inhibit human platelet aggregation, at least partly through the inhibition of PLCγ and Akt, leading to a decrease in TXB2 formation and granule secretion.

  15. Inhibitory Effects of Yuzu and Its Components on Human Platelet Aggregation

    PubMed Central

    Kim, Tae-Ho; Kim, Hye-Min; Park, Se Won; Jung, Yi-Sook

    2015-01-01

    Our previous study demonstrated that yuzu has an anti-platelet effect in rat blood. In the present study, we examined whether the anti-platelet effect of yuzu can be extended to human blood by investigating its ability to inhibit aggregations induced by various agonists in human platelet rich plasma (PRP). This study also investigated the underlying mechanism of yuzu focusing on ADP granule secretion, TXB2 formations, and PLCγ/Akt signaling. The results from this study showed that ethanolic yuzu extract (YE), and its components, hesperidin and naringin, inhibited human platelet aggregation in a concentration-dependent manner. YE, hesperidin and naringin also inhibited TXB2 formation and ADP release. The phosphorylation of PLCγ and Akt was significantly inhibited by YE, heperidin and naringin. Furthermore, we demonstrated that YE, heperidin and naringin has anti-platelet effects in rat ex vivo studies, and lower side effects in mice tail bleeding time studies. The results from this study suggest that YE, hesperidin and naringin can inhibit human platelet aggregation, at least partly through the inhibition of PLCγ and Akt, leading to a decrease in TXB2 formation and granule secretion. PMID:25767683

  16. Computational analysis of platelet adhesion and aggregation under stagnation point flow conditions.

    PubMed

    Reininger, C B; Lasser, R; Rumitz, M; Böger, C; Schweiberer, L

    1999-01-01

    The clinical relevance of platelet function assessment with stagnation point flow adhesio-aggregometry (SPAA) has been verified. Quantitative analysis of platelet adhesion and aggregation is possible by means of mathematical analysis of the dark-field, light intensity curves (growth curves) obtained during the SPAA experiment. We present a computational procedure for evaluating these curves, which was necessitated by, and is based on, actual clinical application. A qualitative growth curve classification, corresponding to a basic and distinct pattern of platelet deposition and characteristic of a regularly occurring clinical state is also presented.

  17. Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation.

    PubMed

    Feys, H B; Pottel, H; Coene, J; Vandewalle, G; Vandekerckhove, P; Compernolle, V

    2016-11-01

    Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history.

  18. Inhibition of bovine platelets aggregation in response to Hyalomma anatolicum salivary gland proteins/peptides

    PubMed Central

    Surbhi; Sangwan, Nirmal; Sangwan, Arun K.; Singh, Vijender; Kumar, Ankit

    2016-01-01

    Aim: Ticks are obligate ectoparasites that have an impact on wide range of vertebrates and also act as a potential vector for the transmission of tropical theileriosis, babesiosis, etc., causing significant loss to livestock production worldwide. While feeding, they introduce their saliva containing different bioactive molecules into the host. These molecules have the capability to counteract the host hemostatic mechanism to suck host blood successfully. Therefore, the study was aimed to isolate anti-platelet aggregating peptides from salivary gland extract (SGE) of Hyalomma anatolicum ticks, a commonly available tick in India. Materials and Methods: Female H. anatolicum salivary glands were dissected out and SGE was prepared by homogenizing it in a suitable buffer under ice. Extract so obtained was fractionated by gel filtration chromatography using Sephacryl S-200 column. Total protein concentration in fractions was estimated and bovine platelets were isolated, stimulated with thrombin (positive control), treated with Gly-Pro-Arg-Pro amide (negative control) and with salivary gland fractions for identification of proteins/peptides having anti-platelet aggregating activities. Results: Proteins/peptides present in various salivary gland fractions inhibited the bovine platelet aggregation and the percent inhibition ranged between 33% and 35.8%. Conclusion: The results suggests that the fractions of H. anatolicum salivary glands possess thrombin-induced anti-platelet aggregating activity and which could be further exploited for raising anti-tick vaccine and also for therapeutic purpose. PMID:27956779

  19. Platelet hemostasis in patients with metabolic syndrome and type 2 diabetes mellitus: cGMP- and NO-dependent mechanisms in the insulin-mediated platelet aggregation.

    PubMed

    Suslova, Tatiana E; Sitozhevskii, Alexei V; Ogurkova, Oksana N; Kravchenko, Elena S; Kologrivova, Irina V; Anfinogenova, Yana; Karpov, Rostislav S

    2014-01-01

    Patients with metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) have high risk of microcirculation complications and microangiopathies. An increase in thrombogenic risk is associated with platelet hyperaggregation, hypercoagulation, and hyperfibrinolysis. Factors leading to platelet activation in MetS and T2DM comprise insulin resistance, hyperglycemia, non-enzymatic glycosylation, oxidative stress, and inflammation. This review discusses the role of nitric oxide (NO) in the regulation of platelet adhesion and aggregation processes. NO is synthesized both in endotheliocytes, smooth muscle cells, macrophages, and platelets. Modification of platelet NO-synthase (NOS) activity in MetS patients can play a central role in the manifestation of platelet hyperactivation. Metabolic changes, accompanying T2DM, can lead to an abnormal NOS expression and activity in platelets. Hyperhomocysteinemia, often accompanying T2DM, is a risk factor for cardiovascular accidents. Homocysteine can reduce NO production by platelets. This review provides data on the insulin effects in platelets. Decrease in a number and sensitivity of the insulin receptors on platelets in T2DM can cause platelet hyperactivation. Various intracellular mechanisms of anti-aggregating insulin effects are discussed. Anti-aggregating effects of insulin are mediated by a NO-induced elevation of cGMP and upregulation of cAMP- and cGMP-dependent pathways. The review presents data suggesting an ability of platelets to synthesize humoral factors stimulating thrombogenesis and inflammation. Proinflammatory cytokines are considered as markers of T2DM and cardiovascular complications and are involved in the development of dyslipidemia and insulin resistance. The article provides an evaluation of NO-mediated signaling pathway in the effects of cytokines on platelet aggregation. The effects of the proinflammatory cytokines on functional activity of platelets are demonstrated.

  20. [Effect of acetylsalicylic acid in complex with lipid nanostructures of various compositions on human platelet aggregation].

    PubMed

    Suslina, Z A; Prokhorov, D I; Shilova, A G; Kaplun, A P; Ionova, V G; Seĭfulla, R D

    2011-01-01

    The effect of lipid nanocomplexes loaded with acetylsalicylic acid (aspirin) on platelet aggregation in vitro was investigated. The antithrombotic effect of aspirin in complex with liposomes prepared from pig brain glycosphingolipids is not only significantly higher compared to control, but also accompanied by leveling of the development of proaggregant effects. It was shown that ADP-induced platelet aggregation is reduced by the introduction of electrostatic charge in the structure of lipid bilayer of liposomes. The effect achieved for the liposomes possessing a negative charge was more pronounced in comparison to the effect of positively charged liposomes.

  1. Inhibitory effects of in vivo oxidized high-density lipoproteins on platelet aggregation: evidence from patients with abetalipoproteinemia.

    PubMed

    Calzada, Catherine; Véricel, Evelyne; Colas, Romain; Guillot, Nicolas; El Khoury, Graziella; Drai, Jocelyne; Sassolas, Agnès; Peretti, Noël; Ponsin, Gabriel; Lagarde, Michel; Moulin, Philippe

    2013-07-01

    There is evidence that high-density lipoproteins (HDLs) may regulate platelet function, but disparate results exist regarding the effects of oxidized HDLs on platelets. The objective of our study was to determine the role of in vivo oxidized HDLs on platelet aggregation. Platelet aggregation and redox status were investigated in 5 patients with abetalipoproteinemia (ABLP) or homozygous hypobetalipoproteinemia, two rare metabolic diseases characterized by the absence of apolipoprotein B-containing lipoproteins, compared to 5 control subjects. Platelets isolated from plasma of patients with ABLP aggregated 4 to 10 times more than control platelets, depending on the agonist. By contrast, no differences in the extent of platelet aggregation were observed between ABLP platelet-rich plasma (PRP) and control PRP, suggesting the presence of a protective factor in ABLP plasma. ABLP HDLs inhibited agonist-induced platelet aggregation by binding to SR-BI, while control HDLs had no effect. On the other hand, lipoprotein-deficient plasma from patients with ABLP did not inhibit platelet aggregation. Severe oxidative stress was evidenced in patients with ABLP. Compared to control HDLs, ABLP HDLs showed a 40% decrease of α-tocopherol and an 11-fold increased malondialdehyde concentration. These results demonstrate that in vivo oxidized HDLs do not lose their antiaggregatory properties despite oxidation.

  2. Structure and mechanism of action of tau aggregation inhibitors

    PubMed Central

    Cisek, Katryna; Cooper, Grace L.; Huseby, Carol J.; Kuret, Jeff

    2015-01-01

    Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer’s disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects. PMID:25387336

  3. Effect of diabetic duration on hemorheological properties and platelet aggregation in streptozotocin-induced diabetic rats

    PubMed Central

    Yeom, Eunseop; Byeon, Hyeokjun; Lee, Sang Joon

    2016-01-01

    Diabetes mellitus with abnormal glucose concentration is associated with changes in hemorheological properties, endothelial function, and platelets hyperactivity. Disturbances may significantly be responsible for diabetes-related vascular complications. In this study, hemorheological and hemodynamic properties were measured according to diabetic duration after streptozotocin treatment in rats. For ex vivo measurements, an extracorporeal model was adopted. Flow rate and blood viscosity were measured using a microfluidic device. Erythrocyte aggregation and morphological parameters of erythrocytes were measured by modified erythrocyte sedimentation rate and the phase-contrast holography under in vitro conditions. The platelet aggregation and mean pressure in the femoral artery were estimated under ex vivo conditions. Hemorheological properties including blood viscosity, erythrocyte aggregation and shape parameters for the control group are significantly different with those for diabetic groups. The changes with respect to diabetic duration were relatively unnoticeable. However, the platelet aggregation is strongly dependent on the diabetic duration. Based on these results, hyperglycemia exposure may induce hemorheological variations in early stages of diabetes mellitus. High platelet aggregation may become more pronounced according to the diabetic duration caused by variations in hemorheological properties resulting in endothelial dysfunction. This study would be helpful in understanding the effects of diabetic duration on biophysical properties. PMID:26898237

  4. Anti-platelet and anti-thrombosis characteristics of Z4A5, a novel selective platelet glycoprotein IIb/IIIa inhibitor, compared with eptifibatide under long-term infusion.

    PubMed

    Shi, X L; Shen, S; Guo, M M; Zhang, G J; Che, J; Wang, B; Zhou, J

    2015-12-01

    Platelet Glycoprotein IIb/IIIa inhibitors are approved for the treatment of acute coronary syndromes and percutaneous coronary interventions due to their effects on the final common pathway of platelet aggregation. Z4A5 is a new hexapeptide IIb/IIIa inhibitor with antiplatelet and antithrombotic effects. This study was performed to assess the characteristics of Z4A5 compared with another IIb/IIIa inhibitor eptifibatide. Light-transmission aggregometry was used to measure platelet aggregation to assess the antiplatelet efficacy of Z4A5 in vitro and ex vivo in beagles. The time course of platelet inhibition and bleeding time prolongation during i.v. bolus plus infusion and after infusion of the Z4A5 were evaluated in beagles following two 2 x 2 Latin square designs. We also compared the antithrombotic activity of Z4A5 with eptifibatide in arterial thrombosis and arteriovenous shunt thrombosis model in beagles. Our data showed that Z4A5 completely inhibited adenosine diphosphate (ADP)-, thrombin- and arachidonic acid-induced in vitro platelet aggregation with values of IC50 of 260 nM, 128.6 and 56.4 n respectively. Z4A5 also markedly and stably prevented ADP-induced ex vivo platelet aggregation and prolonged the bleeding time throughout the 8-hour infusion. Both platelet function and bleeding time returned to normal sooner after cessation of Z4A5 infusion than after eptifibatide. Z4A5 inhibited thrombosis and had the same potent antithrombotic activity as eptifibatide. In conclusion, Z4A5 has the same potent antiplatelet effect and antithrombotic activity with the advantage of a faster on and off time compared to eptifibatide.

  5. Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing.

    PubMed

    Ahmetaj-Shala, B; Tesfai, A; Constantinou, C; Leszczynski, R; Chan, M V; Gashaw, H; Galaris, G; Mazi, S; Warner, T D; Kirkby, N S; Mitchell, J A

    2017-03-18

    Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol(®)) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

  6. Modification of Platelet Proteins by 4-hydroxynonenal: Potential Mechanisms for Inhibition of Aggregation and Metabolism

    PubMed Central

    Ravi, Saranya; Johnson, Michelle S.; Chacko, Balu K.; Kramer, Philip A.; Sawada, Hirotaka; Locy, Morgan L.; Wilson, Landon. S.; Barnes, Stephen; Marques, Marisa B.; Darley-Usmar, Victor M.

    2015-01-01

    Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins. PMID:26475426

  7. Acetaminophen and meloxicam inhibit platelet aggregation and coagulation in blood samples from humans.

    PubMed

    Martini, Angela K; Rodriguez, Cassandra M; Cap, Andrew P; Martini, Wenjun Z; Dubick, Michael A

    2014-12-01

    Acetaminophen (Ace) and meloxicam (Mel) are the two types of analgesic and antipyretic medications. This study investigated the dose responses of acetaminophen and meloxicam on platelet aggregation and coagulation function in human blood samples. Blood samples were collected from six healthy humans and processed to make platelet-adjusted (100 × 10 cells/μl) blood samples. Acetaminophen (Tylenol, Q-PAP, 100 mg/ml) was added at the doses of 0 μg/ml (control), 214 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Similarly, meloxicam (Metacam, 5 mg/ml) was added at doses of 0 μg/ml (control), 2.85 μg/ml (the standard dose, 1 ×), 4 ×, 8 ×, 10 ×, 12 ×, 16 ×, and 20 ×. Fifteen minutes after the addition of acetaminophen and/or meloxicam, platelet aggregation was stimulated with collagen (2 μg/ml) or arachidonic acid (0.5 mmol/l) and assessed using a Chrono-Log 700 aggregometer. Coagulation function was assessed by prothrombin time (PT), activated partial thromboplastin time (aPTT), and using Rotem thrombelastogram. A robust inhibition by acetaminophen and/or meloxicam was observed in arachidonic acid-stimulated platelet aggregation starting at 1 × dose. Collagen-stimulated platelet aggregation was inhibited by ACE starting at 1 × (78 ± 10% of control), and by meloxicam starting at 4 × (72 ± 5% of control, both P < 0.05). The inhibitions by acetaminophen and meloxicam combined were similar to those by acetaminophen or meloxicam. aPTT was prolonged by meloxicam starting at 4 ×. No changes were observed in PT or any of Rotem measurements by acetaminophen and/or meloxicam. Acetaminophen and meloxicam compromised platelet aggregation and aPTT. Further effort is warranted to characterize the effects of acetaminophen and meloxicam on bleeding in vivo.

  8. Analogues of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) as potential anti-platelet-aggregation agents.

    PubMed Central

    Zamecnik, P C; Kim, B; Gao, M J; Taylor, G; Blackburn, G M

    1992-01-01

    Dense granules of platelets contain a high content of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A). We have previously demonstrated an antithrombotic effect of this compound in a live rabbit model. In the present study we find that certain synthetic Ap4A analogues are superior to Ap4A in inhibiting ADP-induced aggregation of human platelets. Analogues having a P--C--P bridge located in the P2,P3 position of Ap4A are the most potent inhibitors. These analogues are also resistant to hydrolytic enzymes. Analogues having the above characteristics exhibit competitive inhibition with ADP in the ADP-induced platelet aggregation reaction. These compounds, such as AppCHFppA, may be useful as antithrombotic agents. The analogues ApSppSpA and ApSpCHFpSpA also showed good inhibitory effects on ADP-induced platelet aggregation. In addition, this action of Ap4A and its analogues provides an example of a dinucleotide inducing an antagonistic effect by occupying an extracellular mononucleotide binding site on platelets. It calls attention to the possibility that Ap4A and its analogues may act in a similar way in whole organisms, triggering effector or inhibitory responses in any one of a variety of cells. PMID:1549600

  9. Bioequivalence and tolerability study of two brands of clopidogrel tablets, using inhibition of platelet aggregation and pharmacodynamic measures☆

    PubMed Central

    Rao, Takallapalli Ramesh Kumar; Usha, P.Rani; Naidu, M.U.R; Gogtay, J.A; Meena, Matthew

    2003-01-01

    Background The role of platelets in acute cardiovascular atherothrombotic events has been well established and attention has focused on platelet inhibition therapy. Clopidogrel is a novel thienopyridine inhibitor of adenosine diphosphate–induced platelet activation. Recent studies have shown that in the setting of coronary angioplasty/stenting, a loading dose of 300 mg followed by 75 mg once daily is required for optimum benefit. Objective This study assessed the bioequivalence and tolerability of 2 oral formulations of clopidogrel 75-mg tablets. Methods This 10-day, open-label, randomized, parallel-group, comparative bioequivalence and tolerability study was carried out in the Department of Clinical Pharmacology and Therapeutics, Nizam's Institute of Medical Sciences (Hyderabad, India). Young healthy male volunteers were enrolled. Subjects were randomized to receive one of two 75-mg tablet formulations of clopidogrel (Clopivas® [test formulation] or Plavix® [reference formulation]). Subjects first received a 300-mg loading dose (four 75-mg tablets) on day 1, followed by 75 mg (1 tablet) at 8:00 AM daily for the next 6 days. Inhibition of platelet aggregation, which is the pharmacologic basis for the therapeutic efficacy of antiplatelet agents, and the effect on bleeding time were used as the pharmacodynamic assessment criteria. Pharmacodynamic variables included mean of maximum activity of percentage of inhibition of platelet aggregation (Emax), mean time to reach Emax (tmax), and mean area under the activity–time curve from time 0 to 168 hours (AUC0–168). Tolerability assessments included blood pressure and heart rate measurements before and at regular intervals (every hour for 12 hours and then at 24 hours) over a 24-hour period after drug administration. Clinical tolerability was assessed using adverse effects, platelet count (assessed on days 3, 6, and 10 after first-dose administration), and neutrophil count (assessed on day 10 after first

  10. 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

  11. Modulation of P-selection and platelet aggregation in chronic periodontitis: A clinical study

    PubMed Central

    Perumal, Ramesh; Rajendran, Maheashwari; Krishnamurthy, Malathi; Ganji, Kiran Kumar; Pendor, Sunil Dattuji

    2014-01-01

    Background: The primary etiologic factor of periodontitis is the subgingival infection with a group of Gram negative pathogens. Transient bacteremia in periodontitis patients underlie chronic production and systemic increases of various proinflammatory mediators, including Interleukin (IL)-1α, IL-6, C-reactive protein and Tumor necrosis factor (TNF)-α. P- selectin is a member of selectin family of cell surface receptor which is located in the membrane of the secretory granules (alpha granules) of platelets and in the membrane of the Weibel-Palade bodies of the vascular endothelial cells. P selectin redistributes from the membrane of the granules to the plasma membrane when platelets and endothelial cells are activated and thus degranulated. Aim: To compare the level of platelet activation, soluble P Selectin level and morphological changes and aggregation of platelets in patients in periodontitis patients compared to healthy controls. Materials and Methods: 80 patients were included in the study with the age group of 35-60. The patients were divided into 2 groups, 40 subjects with generalized chronic periodontitis and 40 healthy subjects taken as control. Periodontal Examination using clinical parameters namely, Bleeding Index, Plaque Index, Probing Pocket Depth and Clinical Attachment Level were recorded. Collection of blood samples for estimation of serum soluble P- selectin level by ELISA method. Evaluation of Platelet morphology and grading the platelet aggregation. Results: P-selectin expression shows that the mean value for control group is 4.97 ± 16.56 ng/mL and study group 13.05 ± 29.94 ng/mL which was significantly higher than control group with P value 0.001. Platelet morphological changes shows small form – mean value for control group is 75.83% ± 14.24% while for study group is 39.08%. ± 21.59; Big form – mean value for control group 0.80% ± 0.35% while for study group 0.48% ± 1.3%and Spider form- mean value for control group 23.88% ± 14

  12. Validity of Particle-Counting Method Using Laser-Light Scattering for Detecting Platelet Aggregation in Diabetic Patients

    NASA Astrophysics Data System (ADS)

    Nakadate, Hiromichi; Sekizuka, Eiichi; Minamitani, Haruyuki

    We aimed to study the validity of a new analytical approach that reflected the phase from platelet activation to the formation of small platelet aggregates. We hoped that this new approach would enable us to use the particle-counting method with laser-light scattering to measure platelet aggregation in healthy controls and in diabetic patients without complications. We measured agonist-induced platelet aggregation for 10 min. Agonist was added to the platelet-rich plasma 1 min after measurement started. We compared the total scattered light intensity from small aggregates over a 10-min period (established analytical approach) and that over a 2-min period from 1 to 3 min after measurement started (new analytical approach). Consequently platelet aggregation in diabetics with HbA1c ≥ 6.5% was significantly greater than in healthy controls by both analytical approaches. However, platelet aggregation in diabetics with HbA1c < 6.5%, i.e. patients in the early stages of diabetes, was significantly greater than in healthy controls only by the new analytical approach, not by the established analytical approach. These results suggest that platelet aggregation as detected by the particle-counting method using laser-light scattering could be applied in clinical examinations by our new analytical approach.

  13. Dauricoside, a new glycosidal alkaloid having an inhibitory activity against blood-platelet aggregation.

    PubMed

    Hu, S M; Xu, S X; Yao, X S; Cui, C B; Tezuka, Y; Kikuchi, T

    1993-10-01

    Dauricoside (1), a new glycosidal alkaloid, was isolated from the rhizomes of Menispermum dauricum DC. along with dauricine (2), daurisoline (3), dauriporphine (4), menisporphine (5), and 6-O-demethylmenisporphine (6), and its structure was determined by means of spectroscopic methods. Compounds 1, 2, and 3 inhibited blood-platelet aggregation induced by adenosine 5'-diphosphate (ADP).

  14. Effect of n-tyrosol on blood viscosity and platelet aggregation.

    PubMed

    Plotnikov, M B; Chernysheva, G A; Smol'yakova, V I; Maslov, M Yu; Cherkashina, I V; Krysin, A P; Sorokina, I V; Tolstikova, T G

    2007-01-01

    Experiments on rats showed that n-tyrosol limited the increase in blood viscosity during thermal exposure at a shear rate of 5-300 sec(-1) and inhibited ADP-induced platelet aggregation. The effects of n-tyrosol are comparable to that of pentoxyphylline.

  15. Uridine Triphosphate Thio Analogues Inhibit Platelet P2Y12 Receptor and Aggregation

    PubMed Central

    Gündüz, Dursun; Tanislav, Christian; Sedding, Daniel; Parahuleva, Mariana; Santoso, Sentot; Troidl, Christian; Hamm, Christian W.; Aslam, Muhammad

    2017-01-01

    Platelet P2Y12 is an important adenosine diphosphate (ADP) receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma, we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 µM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 µM. An eight-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 µM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 µM was 33-fold more effective. A three-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptor was verified by P2Y12 receptor binding and cyclic AMP (cAMP) assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention. PMID:28146050

  16. Reduction of Platelet Aggregation From Ingestion of Oleic and Linoleic Acids Found in Vitis vinifera and Arachis hypogaea Oils.

    PubMed

    Bazán-Salinas, Irma Leticia; Matías-Pérez, Diana; Pérez-Campos, Eduardo; Pérez-Campos Mayoral, Laura; García-Montalvo, Iván Antonio

    The purpose of this study was to evaluate the effect of the consumption of seed oils from Vitis vinifera and Arachis hypogaea in platelet aggregation. The initial hypothesis suggested that subjects who have consumed these seed oils undergo modified platelet aggregation. This study was performed using a pre-post test design, with a control group, and double blind. The effects of the consumption of grape seed and peanut oils were measured for platelet aggregation in clinical and laboratory tests in 30 healthy subjects. In addition to this group, a control group of 4 health subjects received no treatment with oils, just 500 mg oral administration acetylsalicylic acid for 7 days. Platelet aggregation was assessed by the Born turbidimetric method, using 3 different concentrations of adenosine diphosphate as agonists (2, 54; 1, 17; and 0, 58 μM). The study subjects had very similar results; both oils were shown to have a significant reduction in platelet aggregation. Grape seed oil showed a decrease of 8.4 ± 1% in aggregation, compared with peanut oil, which decreased aggregation by 10.4 ± 1%. The control group, taking 500 mg OD aspirin for 7 days, showed a significant decrease in platelet aggregation, similar to that of oil ingestion. Each of the oils was analyzed for fatty acids, to determine which particular acids were presents in greater levels, which could explain the reduction in platelet aggregation. The oil found to be most abundant in grape seeds was linoleic acid (omega-6), and in peanuts, it was oleic acid (omega-9). However, in fact, both acids reduced platelet aggregation. Consumption of plant oils from grape seeds and peanuts had a lowering effect on platelet aggregation, in addition to containing a high content of unsaturated fatty acids. However, omega-3, omega-6, and omega-9 fatty acids were not specifically responsible for the reductions mentioned above.

  17. Effects of heparin on platelet aggregation and release and thromboxane A2 production

    SciTech Connect

    Mohammad, S.F.; Anderson, W.H.; Smith, J.B.; Chuang, H.Y.; Mason, R.G.

    1981-08-01

    Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with /sup 3/H-arachidonic acid and /sup 14/C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of /sup 14/C-serotonin and /sup 3/H-arachidonic acid metabolites, it appeared that either release of /sup 14/C and /sup 3/H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both /sup 14/C and /sup 3/H.

  18. [Recurrent idiopathic cerebral infarction in a 5-year-old boy, with emphasis on the importance of platelet aggregation analysis for appropriate selection of anti-platelet drugs].

    PubMed

    Sugiyama, Nobuyoshi; Matsuda, Shin-ichi; Shimizu, Mie; Obara, Saori; Ikegami, Mariko; Yokoyama, Jyun-ichi; Miyashita, Yoshihiro; Takizawa, Shyunya; Takagi, Shigeharu

    2009-01-01

    We present a 5-year-old boy with recurrent idiopathic cerebral infarction in which analysis of platelet hyperaggregability was useful in choosing appropriate anti-platelet drugs. The patient presented with gait disturbance at the age of 5 years and 1 month. Brain MRI demonstrated multiple infarctions in the right thalamus and left cerebellum. There were no apparent underlying diseases including hematological, cardiac and vascular abnormalities. He was diagnosed as idiopathic cerebral infarction. First, we administered ticlopidine and he remained stable with persistent mild intention tremor in the left upper extremity for 4 months. Then he developed the second stroke at the age of 5 years and 5 months, and multiple infarctions in the right celebellum and cerebellar vermis were demonstrated. On platelet aggregation analysis, adenosine diphosphate (ADP)-induced aggregation was inhibited, probably due to ticlopidine administration. Collagen- and epinephrine-induced platelet aggregation showed hyperaggregation, so we started to administer cilostazol, which inhibits only epinephrine-induced hyperaggregation. We also added aspirin, which inhibits collagen-induced hyperaggregation. The combination of anti-platelet drugs inhibited epinephrine-, collagen- and ADP-induced hyperaggregation in this patient. He has been stable on the triple combination of anti-platelet drugs without further episodes of cerebral infarction or transient ischemic attack for 4 years to date. Appropriate selection of anti-platelet therapy was achieved by the simple and repeatable platelet aggregation analyses, which must be considered even in pediatric patients with cerebral infarction.

  19. Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides

    PubMed Central

    1978-01-01

    The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro. PMID:627836

  20. Warm-up exercise suppresses platelet-eosinophil/neutrophil aggregation and platelet-promoted release of eosinophil/neutrophil oxidant products enhanced by severe exercise in men.

    PubMed

    Wang, Jong-Shyan; Yen, Hsiang-Ling; Yang, Chuen-Mao

    2006-03-01

    Heterotypic platelet-eosinophil/neutrophil aggregation and subsequent release of eosinophil/neutrophil oxidant products contribute to pathogenesis of conditions such as asthma and inflammatory bowel diseases. This study investigates whether warmup exercise (WUE) affects platelet-eosinophil/neutrophil interaction mediated by high-intensity exercise (HIE). Twenty-three healthy sedentary men performed on three occasions light-intensity exercise (LIE, 40%VO(2 max) for 40 min) and HIE (80%VO(2 max) for 40 min) with and without WUE (40%VO(2 max) for 20 min). Before and immediately after exercise, platelet-eosinophil and platelet-neutrophil aggregation (PEA and PNA), reactive oxygen species production of eosinophils and neutrophils (EROS and NROS) enhanced by platelets, and adhesion molecule expression on platelets, eosinophils, and neutrophils were measured. The results of this study demonstrated that HIE enhanced PEA, PNA, and platelet-induced EROS and NROS, was accompanied by increased expressions of Mac-1 on eosinophils and neutrophils and P-selectin on platelets at 5 dyne/cm(2) of shear stress, 100 microg/ml lipopolysaccharide, and 1 microM N-formylmethionyl- leucyl-phenylalanine treatments, whereas the enhancement of HIE on platelet-eosinophil/neutrophil interaction was suppressed by WUE. Conversely, LIE significantly reduced PEA and PNA, suppressed platelet-induced EROS and NROS, and down-regulated eosinophil/neutrophil Mac-1 and platelet P-selectin expressions under various stimuli and shear flow conditions. Moreover, these effects were more pronounced in platelet interaction with eosinophils than with neutrophils. It is concluded that HIE enhances hetero-aggregation, adhesion molecules expressions, and subsequent oxidative bursts mediated by platelets and eosinophils/neutrophils, this effect diminishes after WUE. However, LIE minimizes the risk of thromboinflammation.

  1. Responses to aggregating agents after cleavage of GPIb of human platelets by the O-sialoglycoprotein endoprotease from Pasteurella haemolytica- potential surrogates for Bernard-Soulier platelets?

    PubMed

    Kinlough-Rathbone, R L; Perry, D W; Rand, M L; Packham, M A

    2000-07-15

    Most proteolytic enzymes that cleave glycoprotein lb (GPlb) also cleave other glycoproteins or receptors on the surface of platelets. We have used an O-sialoglycoprotein endoprotease from Pasteurella haemolytica that selectively cleaves the heavily O-glycosylated GPlb, but does not cleave N-linked glycoproteins or unglycosylated proteins. Isolated, [14C]serotonin-labeled platelets in Tyrode-albumin solution were incubated with 10 microg/mL endoprotease for 60 minutes at 37 degrees C. These platelets did not release [14C]serotonin, had no detectable GPIb, and were unresponsive to ristocetin/von Willebrand factor. Compared with control platelets, aggregation and release of [14C]serotonin by the endoprotease-pretreated platelets were inhibited in response to low concentrations of thrombin, SFLLRN (the PAR-1-activating peptide), collagen, and U46619 (a thromboxane A(2) mimetic); aggregates were smaller in size. The presence of fibrinogen overcame the inhibition of responses induced by SFLLRN, collagen, and U46619. With fibrinogen, primary ADP-induced aggregation was scarcely affected by pretreatment with the endoprotease. Thus, the PAR-1 receptor for thrombin, and receptors for collagen, thromboxane A(2), fibrinogen (GPIIb/IIIa), and ADP appear to function normally on the endoprotease-pretreated platelets. Since only GPIb is cleaved by the endoprotease, these platelets seem to provide potential surrogates for Bernard-Soulier syndrome platelets for further studies of platelet functions in this condition.

  2. The effect of naloxone and cyproheptadine on pulmonary platelet trapping, hypotension, and platelet aggregability in traumatized dogs

    SciTech Connect

    Almqvist, P.; Kuenzig, M.; Schwartz, S.I.

    1983-05-01

    Adult respiratory distress syndrome (ARDS) is a serious complication of trauma and sepsis. We have earlier shown naloxone, an opiate antagonist, and cyproheptadine, an antiserotonin drug, to be effective in reducing pulmonary platelet trapping (PPT), which is thought to play an important role in the evolution of ARDS in endotoxin-shocked dogs. Endorphins are implicated as pathophysiologic factors in shock, and serotonin is a possible mediator of their action. The present study shows naloxone and cyproheptadine to be equally effective in protecting against PPT in dogs subjected to trauma, and when naloxone is given before the trauma it also obviates the hypotension associated with trauma. In addition, the naloxone- and cyproheptadine-treated animals did not show the increased platelet aggregability usually seen in traumatized dogs.

  3. Effect of dietary copper on platelet volume and aggregation in men

    SciTech Connect

    Milne, D.B.; Gallagher, S.K. )

    1991-03-15

    A study conducted in the authors' laboratory indicated that mean platelet (PLT) volume (MPV) in rats was affected by dietary copper. They suggested that PLT aggregation may also be affected. Ten men aged 19 to 39 yrs, who lived in a metabolic unit, participated in a study that was divided into four six-week dietary periods in a 2 {times} 2 factorial design with 0.65 and 2.65 mg Cu/d and 0.75 and 2.75 g cystine/d as variables. Plasma copper was not significantly affected by the dietary manipulations, but enzymatically measured ceruloplasmin (Cp) was significantly depressed during low copper intake. Maximum 5 {mu}M ADP-stimulated PLT aggregation in platelet rich plasma was significantly greater during low copper intake than during high Cu intake. Total platelet count and collagen or epinephrine stimulated PLT aggregation were not significantly affected by dietary treatment. Four men who responded to low copper intake with elevated serum cholesterol and depressed specific activity of Cp had significantly elevated MPV and maximum ADP-stimulated PLT aggregation during low Cu intake. The other six men exhibited only increased PLT aggregation during only the low copper, low cystine dietary period. These data indicate that dietary copper is a factor in PLT function in humans.

  4. Platelet factor XIIIa release during platelet aggregation and plasma clot strength measured by thrombelastography in patients with coronary artery disease treated with clopidogrel.

    PubMed

    Kreutz, Rolf P; Owens, Janelle; Lu, Deshun; Nystrom, Perry; Jin, Yan; Kreutz, Yvonne; Desta, Zeruesenay; Flockhart, David A

    2015-01-01

    It has been estimated that up to half of circulating factor XIIIa (FXIIIa) is stored in platelets. The release of FXIIIa from platelets upon stimulation with adenosine diphosphate (ADP) in patients with coronary artery disease treated with dual antiplatelet therapy has not been previously examined. Samples from 96 patients with established coronary artery disease treated with aspirin and clopidogrel were examined. Platelet aggregation was performed by light transmittance aggregometry in platelet-rich plasma (PRP), with platelet-poor plasma (PPP) as reference, and ADP 5 µM as agonist. Kaolin-activated thrombelastography (TEG) was performed in citrate PPP. PRP after aggregation was centrifuged and plasma supernatant (PSN) collected. FXIIIa was measured in PPP and PSN. Platelet aggregation after stimulation with ADP 5 µM resulted in 24% additional FXIIIa release in PSN as compared to PPP (99.3 ± 27 vs. 80.3 ± 24%, p < 0.0001). FXIIIa concentration in PSN correlated with maximal plasma clot strength (TEG-G) (r = 0.48, p < 0.0001), but not in PPP (r = 0.15, p = 0.14). Increasing quartiles of platelet-derived FXIIIa were associated with incrementally higher TEG-G (p = 0.012). FXIIIa release was similar between clopidogrel responders and non-responders (p = 0.18). In summary, platelets treated with aspirin and clopidogrel release a significant amount of FXIIIa upon aggregation by ADP. Platelet-derived FXIIIa may contribute to differences in plasma TEG-G, and thus, in part, provide a mechanistic explanation for high clot strength observed as a consequence of platelet activation. Variability in clopidogrel response does not significantly influence FXIIIa release from platelets.

  5. 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.

  6. Platelet interaction with polymerizing fibrin.

    PubMed

    Niewiarowski, S; Regoeczi, E; Stewart, G J; Senyl, A F; Mustard, J F

    1972-03-01

    Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets. The interaction of polymerizing fibrin with platelets was accompanied by small losses of platelet constituents (serotonin, adenine nucleotides, platelet factor 4, and lactic dehydrogenase). This loss did not appear to be the result of the platelet release reaction. Inhibitors of the release reaction or of adenosine diphosphate (ADP)-induced aggregation did not prevent the interaction of platelets with polymerizing fibrin. Apyrase or prostaglandin E(1) (PGE(1)) reduced the extent of platelet aggregation by polymerizing fibrin, but the amount of protein associated with platelets was slightly increased. The interaction of polymerizing fibrin with platelets was completely inhibited by ethylenediaminetetraacetate (EDTA) or ethylene glycol bis (beta-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA).Fibers formed in solutions of polymerizing fibrin were larger in the presence than in the absence of washed platelets, suggesting that platelets affect fibrin polymerization. The adherence of platelets to polymerizing fibrin may be responsible for the establishment of links between platelets and fibrin in hemostatic plugs and thrombi.

  7. RASA3 is a critical inhibitor of RAP1-dependent platelet activation

    PubMed Central

    Stefanini, Lucia; Paul, David S.; Robledo, Raymond F.; Chan, E. Ricky; Getz, Todd M.; Campbell, Robert A.; Kechele, Daniel O.; Casari, Caterina; Piatt, Raymond; Caron, Kathleen M.; Mackman, Nigel; Weyrich, Andrew S.; Parrott, Matthew C.; Boulaftali, Yacine; Adams, Mark D.; Peters, Luanne L.; Bergmeier, Wolfgang

    2015-01-01

    The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. PMID:25705885

  8. Horse chestnut extract contracts bovine vessels and affects human platelet aggregation through 5-HT(2A) receptors: an in vitro study.

    PubMed

    Felixsson, Emma; Persson, Ingrid A-L; Eriksson, Andreas C; Persson, Karin

    2010-09-01

    Extract from seeds and bark of horse chestnut (Aesculus hippocastanum L) is used as an herbal medicine against chronic venous insufficiency. The effect and mechanism of action on veins, arteries, and platelets are not fully understood. The aim of this study was to investigate the effects and mechanisms of action of horse chestnut on the contraction of bovine mesenteric veins and arteries, and human platelet aggregation. Contraction studies showed that horse chestnut extract dose-dependently contracted both veins and arteries, with the veins being the most sensitive. Contraction of both veins and arteries were significantly inhibited by the 5-HT(2A) receptor antagonist ketanserin. No effect on contraction was seen with the cyclooxygenase inhibitor indomethacin, the alpha(1) receptor antagonist prazosin or the angiotensin AT(1) receptor antagonist saralasin neither in veins nor arteries. ADP-induced human platelet aggregation was significantly reduced by horse chestnut. A further reduction was seen with the extract in the presence of ketanserin. In conclusion, horse chestnut contraction of both veins and arteries is, at least partly, mediated through 5-HT(2A) receptors. Human platelet aggregation is reduced by horse chestnut. The clinical importance of these findings concerning clinical use, possible adverse effects, and drug interactions remains to be investigated.

  9. Inhibition of Platelet Aggregation by the Leaf Extract of Carica papaya During Dengue Infection: An In Vitro Study.

    PubMed

    Chinnappan, Shobia; Ramachandrappa, Vijayakumar Shettikothanuru; Tamilarasu, Kadhiravan; Krishnan, Uma Maheswari; Pillai, Agiesh Kumar Balakrishna; Rajendiran, Soundravally

    2016-04-01

    Dengue cases were reported to undergo platelet activation and thrombocytopenia by a poorly understood mechanism. Recent studies suggested that Carica papaya leaf extract could recover the platelet count in dengue cases. However, no studies have attempted to unravel the mechanism of the plant extract in platelet recovery. Since there are no available drugs to treat dengue and considering the significance of C. papaya in dengue treatment, the current study aimed to evaluate two research questions: First one is to study if the C. papaya leaf extract exerts its action directly on platelets and second one is to understand if the extract can specifically inhibit the platelet aggregation during dengue viral infection. Sixty subjects with dengue positive and 60 healthy subjects were recruited in the study. Platelet-rich plasma (PRP) and platelet-poor plasma were prepared from both the dengue-infected and healthy control blood samples. Effect of the leaf extract obtained from C. papaya leaves was assessed on plasma obtained as well as platelets collected from both healthy and dengue-infected individuals. Platelet aggregation was significantly reduced when leaf extract preincubated with dengue plasma was added into control PRP, whereas no change in aggregation when leaf extract incubated-control plasma was added into control PRP. Upon direct addition of C. papaya leaf extract, both dengue PRP and control PRP showed a significant reduction in platelet aggregation. Within the dengue group, PRP from severe and nonsevere cases showed a significant decrease in aggregation without any difference between them. From the study, it is evident that C. papaya leaf extract can directly act on platelet. The present study, the first of its kind, found that the leaf extract possesses a dengue-specific neutralizing effect on dengue viral-infected plasma that may exert a protective role on platelets.

  10. An in vivo platform for identifying inhibitors of protein aggregation

    PubMed Central

    Mahood, Rachel A.; Jackson, Matthew P.; Revill, Charlotte H.; Foster, Richard J.; Smith, D. Alastair; Ashcroft, Alison E.; Brockwell, David J.; Radford, Sheena E.

    2015-01-01

    Protein aggregation underlies an array of human diseases, yet only one small molecule therapeutic has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, capable of identifying aggregation-prone sequences in the periplasm of Escherichia coli and inhibitors that prevent their aberrant self-assembly. We demonstrate the power of the system using a range of proteins, from small unstructured peptides (islet amyloid polypeptide and amyloid β) to larger, folded immunoglobulin domains. Configured in a 48-well format, the split β-lactamase sensor readily differentiates between aggregation-prone and soluble sequences. Performing the assay in the presence of 109 compounds enabled a rank ordering of inhibition and revealed a new inhibitor of IAPP aggregation. This platform can be applied to both amyloidogenic and other aggregation-prone systems, independent of sequence or size, and can identify small molecules or other factors able to ameliorate or inhibit protein aggregation. PMID:26656088

  11. Chronic lead treatment accelerates photochemically induced platelet aggregation in cerebral microvessels of mice, in vivo

    SciTech Connect

    Al Dhaheri, A.H.; El-Sabban, F.; Fahim, M.A.

    1995-04-01

    Effects of two chronic treatment levels with lead on platelet aggregation in cerebral (pial) microcirculation of the mouse were investigated. Exposure to lead was made by subcutaneous injections for 7 days of lead acetate dissolved in 5% glucose solution, vehicle. Two doses of lead were used, a low dose of 0.1 mg/kg and a high dose of 1.0 mg/kg. Adult male mice were divided into three groups, 10 each; one group was injected with vehicle (control), another was injected with the low dose, and the third was injected with the high dose. Additional mice were used for the determination of hematological parameters and for the lead level in serum of the three groups. On the eighth day, platelet aggregation in pial microvessels of these groups of mice was carried out in vivo. Animals were anesthetized (urethane, 1-2 mg/g, ip), the trachea was intubated, and a craniotomy was performed. Platelet aggregation in pial microvessels was induced photochemically, by activation of circulating sodium fluorescein (0.1 mg/25 g, iv) with an intense mercury light. The time required for the first platelet aggregate to appear in pial arterioles was significantly shorter in the lead-treated mice than in control. This effect was in a dose-dependent manner; 113 {+-} 44 sec for low dose and 71 {+-} 18 sec for high dose vs 155 {+-} 25 sec for control, P < 0.02 and P < 0.001, respectively. Between the two lead-treated groups, the high dose significantly (P < 0.05) shortened the time to first aggregate. These data evidenced an increased susceptibility to cerebrovascular thrombosis as a result of exposure to lead. 26 refs., 4 figs., 2 tabs.

  12. Effect of diltiazem and low-dose aspirin on platelet aggregation and ATP release induced by paired agonists.

    PubMed

    Zucker, M L; Budd, S E; Dollar, L E; Chernoff, S B; Altman, R

    1993-08-02

    The authors studied the effects of diltiazem, administered alone and together with low-dose aspirin, on the platelet response to paired agonists. After a baseline period, 25 healthy volunteers were given oral diltiazem for 1 week (120, 240, or 360 mg/day), and then crossed over randomly between 1 week on diltiazem plus aspirin (81 mg/day), and 1 week on aspirin (81 mg/day) alone. Platelet function was tested on 2 consecutive days in each period. Synergistic platelet aggregation and ATP release were obtained at baseline using a subthreshold concentration of arachidonic acid combined with platelet activating factor, ADP, or epinephrine. Diltiazem resulted in significant decrease from baseline in platelet aggregation and ATP release using the arachidonic acid-epinephrine combination (35% and 40% decrease, respectively, p < 0.01) and a significant decrease in aggregation using the arachidonic acid-ADP combination (22% decrease, p < 0.01). The effects were neither dose-related, nor accompanied by any significant change in serum thromboxane B2 levels or bleeding times. There was no significant difference between the effects of aspirin alone and aspirin plus diltiazem on the synergistic platelet aggregation and ATP release induced by the paired agonists, or on thromboxane B2 levels or bleeding times. Diltiazem administered in vivo partially inhibits the synergistic platelet aggregation and ATP release induced by paired agonists; however, in contrast to a previous in vitro study it does not potentiate the platelet-inhibitory effect of aspirin.

  13. Effects of ethanol on aggregation, serotonin release, and amyloid precursor protein processing in rat and human platelets.

    PubMed

    Ehrlich, Daniela; Humpel, Christian

    2014-01-01

    It is known that oxidative stress leads to amyloid precursor protein (APP) dysregulation in platelets. Ethanol (EtOH) is a vascular risk factor and induces oxidative stress. The aim of the present study was thus to investigate whether EtOH affects APP processing in rat and human platelets. Platelets were exposed to 50 mM EtOH with and without 2 mM calcium-chloride (CaCl₂) for 20 or 180 minutes at 37°C. Platelet aggregation, serotonin release and APP isoforms 130 and 106/110 kDa were analyzed. As a control, 100 mM H₂O₂ was tested in rat platelets. Our data show that EtOH alone did not affect any of the analyzed parameters, whereas CaCl₂ significantly increased aggregation of rat and human platelets. In addition, CaCl₂ alone enhanced serotonin release in rat platelets. EtOH counteracted CaCl₂-induced aggregation and serotonin release. In the presence of CaCl₂, EtOH reduced the 130 kDa APP isoform in rat and human platelets. In conclusion, this study shows that in the presence of CaCl₂, EtOH affects the platelet function and APP processing in rat and human platelets.

  14. Endotoxin Interactions with Platelets

    DTIC Science & Technology

    1985-01-01

    irreversible aggregation of human platelets (Hamberg and Sainuelsson 1974; Hlamberg et al 1975). Acetylsalicylic acid , an inhibitor of cyclooxygenase aud...exposure to endotoxin (100 ttg/nil). To simulate the lipopolysac- charide of endotoxin, several different fatty acids were added individually to platelet...platelet lytic capability. Similarity, iflegaradt doses of ganima radiation 6wCo destroy fatty acid groups on lipid A (L. Bertok, personal communication

  15. Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment.

    PubMed

    Miyata, Kenichi; Takagi, Satoshi; Sato, Shigeo; Morioka, Hiroshi; Shiba, Kiyotaka; Minamisawa, Tamiko; Takami, Miho; Fujita, Naoya

    2014-12-01

    Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus. Aggrus was observed to be identical to the marker protein podoplanin (alternative names, T1α, OTS-8, and others). Aggrus is frequently overexpressed in several types of tumors and enhances platelet aggregation by interacting with the platelet receptor C-type lectin-like receptor 2 (CLEC-2). Here, we generated a novel single-chain antibody variable region fragment (scFv) by linking the variable regions of heavy and light chains of the neutralizing anti-human Aggrus monoclonal antibody MS-1 with a flexible peptide linker. Unfortunately, the generated KM10 scFv failed to suppress Aggrus-induced platelet aggregation in vitro. Therefore, we performed phage display screening and finally obtained a high-affinity scFv, K-11. K-11 scFv was able to suppress Aggrus-induced platelet aggregation in vitro. Moreover, K-11 scFv prevented the formation of pulmonary metastasis in vivo. These results suggest that K-11 scFv may be useful as metastasis inhibitory scFv and is expected to aid in the development of preclinical and clinical examinations of Aggrus-targeted cancer therapies.

  16. Dietary calcium attenuates platelet aggregation and intracellular Ca2+ mobilization in spontaneously hypertensive rats

    NASA Technical Reports Server (NTRS)

    Otsuka, K.; Watanabe, M.; Yue, Q.; McCarron, D. A.; Hatton, D.

    1997-01-01

    Spontaneously hypertensive rats (SHR) are known to be blood pressure sensitive to dietary calcium. The effects of dietary calcium on platelet aggregation and intracellular Ca2+ mobilization were assessed by turbidimetric methods and fura-2 methods, respectively, in washed platelets of SHR. Ca2+ ATPase activity was examined in aortic membrane fractions. Six weeks of dietary calcium supplementation attenuated the increase of systolic blood pressure (SBP 199 +/- 16 v 170 +/- 9 mm Hg, P < .001) and thrombin-induced platelet aggregation (84.5 +/- 3.7 v 73.7 +/- 7.4%, P < .004) at 9 weeks of age. The ionomycin-induced intracellular calcium ([Ca2+]i) peak in the absence of external Ca2+, which reflects [Ca2+]i storage size, and thrombin-evoked [Ca2+]i release from [Ca2+]i storage were decreased by 2.0% Ca diet (472 +/- 55 v 370 +/- 23 nmol/L, P < .001, 339 +/- 29 v 278 +/- 33 nmol/L, P < .002). In addition, SBP was positively correlated with platelet aggregation (r = 0.703, P = .0088), thrombin-evoked [Ca2+]i (r = 0.739, P = .0044), and ionomycin-induced [Ca2+]i (r = 0.591, P = .0415), respectively. However, there was no significant effect of dietary calcium on Ca2+-ATPase activity in aortic membranes. These results suggest that dietary calcium supplementation had a beneficial effect on platelets of SHR by attenuating [Ca2+]i mobilization from [Ca2+]i storage. The hypotensive effect of dietary calcium might be associated with attenuated [Ca2+]i mobilization in SHR.

  17. Prevention of platelet glycoprotein IIb/IIIa activation by 3,4-methylenedioxy-beta-nitrostyrene, a novel tyrosine kinase inhibitor.

    PubMed

    Wang, Wei-Ya; Wu, Yang-Chang; Wu, Chin-Chung

    2006-10-01

    Binding fibrinogen to activated glycoprotein (GP)IIb/IIIa is the final common pathway of platelet aggregation and has become a successful target for antiplatelet therapy. In the present study, we found that a small chemical compound, 3,4-methyl-enedioxy-beta-nitrostyrene (MNS), exhibited potent and broad-spectrum inhibitory effects on human platelet aggregation caused by various stimulators. Moreover, addition of MNS to human platelets that had been aggregated by ADP caused a rapid disaggregation. We demonstrated that the antiaggregatory activity of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. In contrast, MNS is not a direct antagonist of GPIIb/IIIa, because MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets. By investigating how MNS inhibits GPIIb/IIIa activation, we found that MNS potently inhibited the activity of tyrosine kinases (Src and Syk) and prevented protein tyrosine phosphorylation and cytoskeletal association of GPIIb/IIIa and talin, but it had no direct effects on protein kinase C, Ca2+ mobilization, Ca2+-dependent enzymes (myosin light chain kinase and calpain), and arachidonic acid metabolism, and it did not affect the cellular levels of cyclic nucleotides. Therefore, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation without directly affecting other signaling pathways required for platelet activation. Because MNS inhibits GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists, this feature may provide a new strategy for treatment of platelet-dependent thrombosis.

  18. Anti-platelet aggregation triterpene saponins from the galls of Sapindus mukorossi.

    PubMed

    Huang, Hui-Chi; Tsai, Wei-Jern; Liaw, Chia-Ching; Wu, Shih-Hsiung; Wu, Yang-Chang; Kuo, Yao-Haur

    2007-09-01

    Bioassay-directed fractionation of an ethanolic extract of the galls of Sapindus mukorossi has resulted in the isolation of two new tirucallane-type triterpenoid saponins, sapinmusaponins Q (1) and R (2), along with three known oleanane-type triterpenoid saponins (3-5). Their structures were elucidated on the basis of spectroscopic analysis and chemical hydrolysis. Biological evaluation showed that both sapinmusaponins Q and R demonstrated more potent anti-platelet aggregation activity than aspirin.

  19. Coherent Plasmon-Exciton Coupling in Silver Platelet-J-aggregate Nanocomposites

    DTIC Science & Technology

    2015-02-27

    plexcitons, which may result in unusual light-matter interactions. We report the formation of a transparency dip in the visible spectra of colloidal ...visible spectra of colloidal suspensions containing silver nanoplatelets and a cyanine dye, 1,1?-diethyl-2,2?-cyanine iodide (PIC). PIC was...highest reported for colloidal nanoparticles. The optical properties of the silver platelet-J-aggregate nanocomposites were supported numerically and

  20. Effects of ergotamine and methysergide on blood platelet aggregation responses of migrainous subjects

    PubMed Central

    Hilton, Barbara P.; Zilkha, K. J.

    1974-01-01

    Platelet aggregation responses to 5-hydroxytryptamine (5-HT) were measured in plasma from migrainous subjects taking either methysergide maleate or ergotamine tartrate and were found to be reduced. Blood 5-HT levels of subjects free of headache were not affected by these drugs. The results support the hypothesis that methysergide and ergotamine act by occupying 5-HT uptake sites in vessel walls, leaving 5-HT molecules available to occupy receptors concerned with vasoconstriction. PMID:4836755

  1. The influence of Rubus idaeus and Rubus caesius leaf extracts on platelet aggregation in whole blood. Cross-talk of platelets and neutrophils.

    PubMed

    Dudzinska, Dominika; Bednarska, Katarzyna; Boncler, Magdalena; Luzak, Boguslawa; Watala, Cezary

    2016-07-01

    Recently, polyphenols have gained attention as potential natural cardioprotective therapeutics, due to their antiplatelet, anti-inflammatory and anticoagulant activity. Species belonging to the genus Rubus sp. have been reported to be a source of polyphenolic compounds with antioxidative proprieties and beneficial biological activities. This study investigates the effects of leaf extracts obtained from red raspberry (Rubus idaeus L.) and European dewberry (Rubus caesius L.) on the reactivity of blood platelets. In ADP-stimulated blood, raspberry and dewberry extracts (15 µg/ml) markedly decreased platelet surface membrane expression of activated GPIIbIIIa receptor by 16% and 21%, respectively (P < 0.01) and significantly inhibited platelet aggregation (by 31-41% for raspberry and by 38-55% for dewberry, P < 0.01). In platelet-rich plasma (PRP), the extracts had no effect on ADP-induced platelet aggregation. The effectiveness of the extracts in whole blood and the lack of their activity in PRP indicate that leukocytes are likely to participate in the platelet response to the extracts. Our experiments show that the extracts significantly reduced the amount of free radicals released by activated neutrophils in whole blood (P < 0.001), as well as in suspensions of isolated neutrophils (P < 0.05). Moreover, the reduced number of neutrophils leads to the decreased efficiency of the extracts in the inhibition of platelet aggregation. In summary, our findings show that the raspberry and dewberry leaf extracts considerably modulated blood platelet reactivity in whole blood: they influenced blood platelet aggregation, possibly via the modulation of the redox status dependent on the oxidative activity of neutrophils.

  2. Release of Phosphorylated HSP27 (HSPB1) from Platelets Is Accompanied with the Acceleration of Aggregation in Diabetic Patients.

    PubMed

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Enomoto, Yukiko; Matsushima-Nishiwaki, Rie; Onuma, Takashi; Kojima, Akiko; Doi, Tomoaki; Tanabe, Kumiko; Akamatsu, Shigeru; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Iwama, Toru; Tanikawa, Takahisa; Ishikawa, Kei; Kojima, Kumi; Kozawa, Osamu

    2015-01-01

    We investigated the relationship between HSP27 phosphorylation and collagen-stimulated activation of platelets in patients with diabetes mellitus (DM). Platelet-rich plasma was prepared from blood of type 2 DM patients. The platelet aggregation was analyzed in size of aggregates by an aggregometer using a laser scattering method. The protein phosphorylation was analyzed by Western blotting. Phosphorylated-HSP27 and PDGF-AB released from platelets were measured by ELISA. The phosphorylated-HSP27 levels at Ser-78 and Ser-82 induced by collagen were directly proportional to the platelet aggregation. Total HSP27 levels in platelets were decreased concomitantly with the phosphorylation. The released HSP27 levels were significantly correlated with the phosphorylated levels of HSP27 in the platelets stimulated by 0.3 μg/ml collagen. The low dose collagen-stimulated release of HSP27 was detected but relatively small in healthy donors. The released levels of PDGF-AB were in parallel with the levels of released HSP27. Area under the curve (AUC) of small aggregation (9-25 μm) induced by 0.3 μg/ml collagen was inversely proportional to the levels of released HSP27. AUC of large aggregation (50-70 μm) was directly proportional to the levels of released HSP27. Exogenous recombinant phosphorylated- HSP27 hardly affected the aggregation or the released levels of PDGF-AB induced by collagen. These results strongly suggest that HSP27 is released from human platelets accompanied with its phosphorylation induced by collagen, which is correlated with the acceleration of platelet aggregation in type 2 DM patients.

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

    SciTech Connect

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

    1989-07-01

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

  4. Inability of probiotic bacterial strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 to induce human platelet aggregation in vitro.

    PubMed

    Zhou, J S; Rutherfurd, K J; Gill, H S

    2005-11-01

    Platelet aggregation contributes to the pathogenesis of infective endocarditis, and aggregation of platelets induced by lactobacilli is thought to be an important contributory factor in the development and progression of Lactobacillus endocarditis. The main purpose of this study was to examine the effect of immunity-enhancing probiotic strains Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019 on the activation and aggregation of human blood platelets. Whole blood samples from healthy individuals were incubated in vitro with HN001 or HN019 and subsequently labeled with platelet-specific monoclonal antibodies, fluorescein isothiocyanate-conjugated anti-CD41a (expressed on normal platelets), and phycoerythrin-streptavidin-conjugated anti-CD62p (expressed on activated platelets) before analysis by flow cytometry. Platelet-rich plasma was used to assist the gating of the platelet cluster. ADP and epinephrine were used as the physiological platelet activation agonists. Platelet aggregation-inducing strain Streptococcus sanguis 133-79 was used as a positive control strain. The mean fluorescence intensity of phycoerythrin and the percentage of platelets expressing the CD62p marker were used to assess the degree of platelet activation. The percentage of CD62p-positive platelets and the light scatter profiles of the agonist-activated platelets were used to identify the occurrence and degree of platelet aggregation. HN001 and HN019 had no effect on spontaneous platelet activation and aggregation; they also failed to exacerbate the platelet aggregation activity induced by ADP and epinephrine. Therefore, these test probiotic strains HN001 and HN019 are less likely to participate in the pathogenesis of infective endocarditis or other thrombotic disorders with regard to platelet aggregation factors.

  5. Effect of the crude extract of Cestrum parqui on carrageenin-induced rat paw oedema and aggregation of human blood platelets.

    PubMed

    Shehnaz, D; Hamid, F; Baqai, F T; Uddin Ahmad, V

    1999-08-01

    An extract of Cestrum parqui aerial parts in methanol:water (1:1) showed inhibition of carrageenin-induced oedema. The aggregation of human blood platelets induced by adenosine diphosphate and platelet activating factor was also inhibited (IC(50)s were 3 and 2 mg/mL, respectively). On the contrary, the extract did not inhibit arachidonic acid-mediated platelet aggregation.

  6. Naturally occurring polyphenolic inhibitors of amyloid beta aggregation.

    PubMed

    Churches, Quentin I; Caine, Joanne; Cavanagh, Kate; Epa, Vidana Chandana; Waddington, Lynne; Tranberg, C Elisabet; Meyer, Adam G; Varghese, Jose N; Streltsov, Victor; Duggan, Peter J

    2014-07-15

    Alzheimer's disease is the most common neurodegenerative disease and is one of the main causes of death in developed countries. Consumption of foods rich in polyphenolics is strongly correlated with reduced incidence of Alzheimer's disease. Our study has investigated the biological activity of previously untested polyphenolic compounds in preventing amyloid β aggregation. The anti-aggregatory potential of these compounds was assessed using the Thioflavin-T assay, transmission electron microscopy, dynamic light scattering and size exclusion chromatography. Two structurally related compounds, luteolin and transilitin were identified as potent inhibitors of Aβ fibril formation. Computational docking studies with an X-ray derived oligomeric structure offer a rationale for the inhibitory activity observed and may facilitate development of improved inhibitors of Aβ aggregation and toxicity.

  7. Platelet response to increased aspirin dose in patients with persistent platelet aggregation while treated with aspirin 81 mg.

    PubMed

    Gengo, Fran; Westphal, Erica S; Rainka, Michelle M; Janda, Maria; Robson, Matthew J; Hourihane, J Maurice; Bates, Vernice

    2016-04-01

    This study demonstrates that patients who are taking 81 mg of aspirin and are nonresponsive benefit from a dose of 162 mg or greater vs a different antiplatelet therapy. We identified 100 patients who were nonresponsive to aspirin 81 mg via whole blood aggregometry and observed how many patients became responsive at a dose of 162 mg or greater. Platelet nonresponsiveness was defined as >10 Ω of resistance to collagen 1 µg/mL and/or an ohms ratio of collagen 1 µg/mL to collagen 5 µg/mL >0.5 and/or >6 Ω to arachidonate. Borderline response was defined as an improvement in 1 but not both of the above criteria. Of the initial 100 patients who were nonresponsive to an aspirin dose of 81 mg, 79% became responsive at a dose of 162 mg or >162 mg. Only 6% did not respond to any increase in dose. We believe that patients treated with low-dose aspirin who have significant risk for secondary vascular events should be individually assessed to determine their antiplatelet response. Those found to have persistent platelet aggregation despite treatment with 81 mg of aspirin have a higher likelihood of obtaining an adequate antiplatelet response at a higher aspirin dose.

  8. Treatment of experimental furcation perforations with mineral trioxide aggregate, platelet rich plasma or platelet rich fibrin in dogs' teeth.

    PubMed

    Tawfik, Hosam E; Abu-Seida, Ashraf M; Hashem, Ahmed A; El-Khawlani, Mohammed M

    2016-06-01

    This work evaluates the effect of mineral trioxide aggregate (MTA), platelet rich plasma (PRP) or platelet rich fibrin (PRF) on healing of non-contaminated and contaminated furcation perforations. A total of 192 teeth of 12 dogs was divided into three equal groups according to evaluation period. Each group was further subdivided into MTA, PRP, PRF, negative and positive control subgroups. Each experimental subgroup was further subdivided according to perforation status into non-contaminated and contaminated subdivisions. Root canal therapy was carried out and furcation perforation was made in all teeth except in negative control subgroup. The furcation perforation was repaired immediately in subdivision (1) and after 4 weeks in subdivision (2). The change in vertical bone loss was measured by radiography. Inflammatory cell count, cemental deposition, new bone formation, bone resorption and epithelial proliferation were assessed. Both PRP and PRF demonstrated statistically significant reduction in vertical bone loss and inflammatory cell count than MTA. No significant difference was found between MTA, PRP and PRF in cemental deposition, new bone formation, bone resorption and epithelial proliferation. The non-contaminated teeth demonstrated better treatment outcomes than the contaminated teeth. In conclusion, PRP and PRF are successful treatment options for repairing of furcation perforation in both non-contaminated and contaminated teeth in dogs with superior outcomes in non contaminated teeth.

  9. Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis

    PubMed Central

    Chang, Yu-Tsui; Lu, Meng-Hong; Huang, Tur-Fu; Chong, Kowit-Yu; Liao, Hsiang-Ruei; Cheng, Ju-Chien; Tseng, Ching-Ping

    2015-01-01

    Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective. PMID:26528756

  10. The effects of 7.5% NaCl/6% dextran 70 on coagulation and platelet aggregation in humans

    NASA Technical Reports Server (NTRS)

    Hess, J. R.; Dubick, M. A.; Summary, J. J.; Bangal, N. R.; Wade, C. E.

    1992-01-01

    The combination solution of 7.5% NaCl/6% dextran 70 (HSD) administered IV gives hemodynamic improvement in the treatment of hemorrhagic hypotension. Since earlier dextran solutions were reported to interfere with blood coagulation, the effects of HSD on the prothrombin time (PT), the activated partial thromboplastin time (APTT), platelet aggregation, and platelet concentration were studied. The HSD mixed with human plasma (1:5 and 1:10) slightly prolonged PT, but had no effect on the APTT, compared with saline controls. The HSD also decreased human platelet aggregation at the 1:5 dilution. In separate mixing studies, the hypertonic saline component of HSD was associated with the prolongation of PT and decreased platelet aggregation. The data from these studies indicate that at its proposed therapeutic dose, HSD is expected to have minimal effect on blood coagulation.

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

    PubMed

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

    2016-06-01

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

  12. [The effects of ticlopidine and nifedipine on platelet aggregation in patients with obliterating arteriopathy of the lower limbs].

    PubMed

    Hevia Alonso, A; Gago Angelino, J; López-Valpuesta, F J; Serrano Molina, J S; Fernández-Sanz, S

    1992-04-01

    This paper evaluates the antiaggregant action of ticlopidine and nifedipine in patients with obliterate arteriopathy in inferior limbs of arteriosclerotic etiology (OAIL). They were established 4 study groups: control, with health volunteers without treatment (N = 10); patients treated with placebo (n = 11); patients treated with 500 mg/day of ticlopidine (n = 12) and treated with 30 mg/day of nifedipine (n = 12). The last 3 groups the treatment duration was 30 days. It was studied the platelet aggregatory activity against ADP and collagen, before drug administration and at 15 and 30 days post-treatment. Our results suggest that: Platelet aggregation is increased in patients with AOMI compared with that observed in the control group. Ticlopidine inhibits platelet aggregation induced by ADP, but not that induced by collagen. Nifedipine doesn't produce any effect on platelet aggregation.

  13. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of /sup 125/I-fibrinogen to rat platelets

    SciTech Connect

    McGregor, L.; Toor, B.; McGregor, J.L.; Renaud, S.; Clemetson, K.J.

    1984-03-01

    The aggregation to ADP and the binding of /sup 125/I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.

  14. Hydroxyapatite formed on/in agarose gel induces activation of blood coagulation and platelets aggregation.

    PubMed

    Arimura, Shin-ichiro; Kawahara, Ko-ichi; Biswas, Kamal Krishna; Abeyama, Kazuhiro; Tabata, Masashi; Shimoda, Toru; Ogomi, Daisuke; Matsusaki, Michiya; Kato, Shinya; Ito, Takashi; Sugihara, Kazumasa; Akashi, Mitsuru; Hashiguchi, Teruto; Maruyama, Ikuro

    2007-05-01

    We reported earlier that hydroxyapatite (HA) formed on/in agarose gels (HA/agarose) produced by alternate soaking process is a bone-filling material possessing osteoconductive and hemostatic effects. This process could allow us to make bone-like apatite that was formed on/in organic polymer hydrogel matrices. Here, we investigated the mechanism of hemostasis induced by HA/agarose and found that HA/agarose, but not agarose or HA powder, significantly shortened activated partial thromboplastin time (APTT). While HA/agarose did not show significant platelet aggregation, it markedly enhanced adenosine diphosphate (ADP)-induced platelet aggregation. Moreover, Western blot analysis revealed selective adsorption of vitronectin onto HA/agarose. We also observed marked differences between HA powder and HA/agarose in their XRD patterns. The crystallinity of HA powder was much higher compared to that of HA/agarose. Furthermore, 50-100 nm of tube-form aggregations was observed in HA powder on the other hand 100-200 nm of particles was observed in HA/agarose by SEM observation. Thus 100-200 nm of low crystallized particles on the surface structure of HA/agarose may play an important role in hemostasis. Our results demonstrated a crucial role of HA/agarose in the mechanism of hemostasis and suggested a potential role for HA/agarose as a bone-grafting material.

  15. Investigation of piperidine derivatives in ex vivo models of pain and platelet aggregation.

    PubMed

    Saify, Zafar Saeed; Rasheed, Huma; Mushtaq, Nousheen; Nisa, Mehrun; Haider, Shazia; Naz, Afshan; Azhar, Kaniz Fizza; Miana, Ghulam Abbas

    2012-11-01

    Piperidine derivatives are known to exhibit analgesic activities and are likely to possess the ability to block the effects of prostaglandins through inhibition of downstream signaling pathways. The present study investigated the activity of five derivatives (PD2-6) of 4-(4'-bromophenyl)-4-piperidinol (PD1), against pain and platelet aggregation mediated by the release of prostaglandins and thromboxane A2, respectively. The results showed that compound PD1 and its two phenacyl derivatives PD3 and PD5 exhibited a highly significant analgesic effect (p < 0.01), whereas PD4 and PD6 also showed significant activity. PD3, the most active analgesic compound when docked to the opioid receptor, had interactions between the oxygen of its nitro group and the amino group of ARG 573, indicating a distance of 1.2563 Å. The antiplatelet data showed that compound PD5 (4-(4'-bromo-phenyl)-4-hydroxy-1-[2-(2″,4″-dimethoxyphenyl)-2-oxo-ethyl]-piperidinium bromide) had an IC(50) = 0.06 mM, which was the most active compound, whereas PD3 was the second most active compound against platelet aggregating factor-induced aggregation with an IC(50) = 80 mM. Acetyl salicylic acid (IC(50) = 150 μM) was used as a positive control.

  16. Detection of Platelet-Monocyte Aggregates by the ADAM® Image Cytometer

    PubMed Central

    Jung, Bo Kyeung; Cho, Chi Hyun; Moon, Kyung Chul; sung Hur, Dae; Yoon, Jeong-Ah; Yoon, Soo-Young

    2014-01-01

    Background: Inappropriate platelet activation is known to be associated with various thrombotic disorders. Platelet-monocyte aggregates (PMAs), whose formation is mediated by platelet surface P-selectin (CD62P), can be used as a reliable marker to detect platelet activation. Previous studies have generally detected PMAs through flow cytometry-based approaches. Recently, the ADAM® image cytometer (Nanoentek Inc., Seoul, Korea) was developed for image-based cellular analysis. In this study, we detected PMAs with the ADAM® cytometer, evaluated the reproducibility of the measurements made by the ADAM® cytometer, and compared the abilities of the ADAM® cytometer and a flow cytometric assay to detect PMAs. Methods: Whole blood samples were collected from patients. Within 5 minutes of collection, anticoagulated whole blood samples were fixed in 10% paraformaldehyde and 5% glyoxal. Nineteen clinical specimens were collected; each was analyzed three times with the ADAM® cytometer in order to assess the reproducibility of its measurements. To compare the ability of the ADAM® cytometer with that of a flow cytometer to detect PMAs, each cytometer was used for 23 clinical samples and the correlation of the measurements was determined. Results: The PMA measurements made by the ADAM® cytometer showed good reproducibility (CV < 10% for all specimens). Moreover, the PMA measurements made by the ADAM® cytometer exhibited a high correlation with those made by a flow cytometric assay (R = 0.944). Conclusions: The ADAM® cytometer is a suitable alternative method to the flow cytometry-based assays. Since the ADAM cytometer does not need specialized instrument knowledge or software proficiency (unlike flow cytometry), the ADAM® cytometer can be used as a rapid and reliable POCT device to measure platelet activation in peripheral blood. This, in turn, will provide valuable information regarding patient propensities to thrombotic diseases. PMID:25317068

  17. Structure, Sulfatide Binding Properties, and Inhibition of Platelet Aggregation by a Disabled-2 Protein-derived Peptide*

    PubMed Central

    Xiao, Shuyan; Charonko, John J.; Fu, Xiangping; Salmanzadeh, Alireza; Davalos, Rafael V.; Vlachos, Pavlos P.; Finkielstein, Carla V.; Capelluto, Daniel G. S.

    2012-01-01

    Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for αIIbβ3 integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2-derived peptide encompassing the sulfatide-binding motifs has been determined in dodecylphosphocholine micelles using NMR spectroscopy. Dab2 sulfatide-binding motif contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes. PMID:22977233

  18. Using bacterial inclusion bodies to screen for amyloid aggregation inhibitors

    PubMed Central

    2012-01-01

    Background The amyloid-β peptide (Aβ42) is the main component of the inter-neuronal amyloid plaques characteristic of Alzheimer's disease (AD). The mechanism by which Aβ42 and other amyloid peptides assemble into insoluble neurotoxic deposits is still not completely understood and multiple factors have been reported to trigger their formation. In particular, the presence of endogenous metal ions has been linked to the pathogenesis of AD and other neurodegenerative disorders. Results Here we describe a rapid and high-throughput screening method to identify molecules able to modulate amyloid aggregation. The approach exploits the inclusion bodies (IBs) formed by Aβ42 when expressed in bacteria. We have shown previously that these aggregates retain amyloid structural and functional properties. In the present work, we demonstrate that their in vitro refolding is selectively sensitive to the presence of aggregation-promoting metal ions, allowing the detection of inhibitors of metal-promoted amyloid aggregation with potential therapeutic interest. Conclusions Because IBs can be produced at high levels and easily purified, the method overcomes one of the main limitations in screens to detect amyloid modulators: the use of expensive and usually highly insoluble synthetic peptides. PMID:22553999

  19. Recombinant albumins containing additional peptide sequences smaller than barbourin retain the ability of barbourin-albumin to inhibit platelet aggregation.

    PubMed

    Sheffield, William P; Wilson, Brianna; Eltringham-Smith, Louise J; Gataiance, Sharon; Bhakta, Varsha

    2005-05-01

    The previously described fusion protein BLAH(6) (Marques JA et al.,Thromb Haemost 2001; 86: 902-8) is a recombinant protein that combines the small disintegrin barbourin with hexahistidine-tagged rabbit serumalbumin (RSA) produced in Pichia pastoris yeast. We sought to determine: (1) if BLAH(6) was immunogenic; and (2) if its barbourin domain could be productively replaced with smaller peptides. Purified BLAH(6) was injected into rabbits, and anti-barbourin antibodies were universally detected in plasma 28 days later; BLAH(6) was, however, equally effective in reducing platelet aggregation in both naive and pre-treated rabbits. Thrombocytopenia was not observed, and complexing BLAH(6) to alpha(IIb)beta(3) had no effect on antibody detection. The barbourin moiety of BLAH(6) was replaced with each of four sequences: Pep I (VCKGDWPC); PepII (VCRGDWPC); PepIII (bar-bourin 41-54); and PepIV (LPSPGDWR). The corresponding fusion proteins were tested for their ability to inhibit ADP-induced platelet aggregation. PepIII-LAH(6) inhibited neither rabbit nor human platelets. PepI-LAH(6) and PepIV-LAH(6) inhibited rabbit platelet aggregation as effectively as BLAH(6), but PepIV-LAH(6) did not inhibit human platelet aggregation. PepI-LAH(6) and PepIILAH(6) inhibited human platelet aggregation with IC(50)s 10- and 20-fold higher than BLAH(6). Cross-immunoprecipitation assays with human platelet lysates confirmed that all proteins and peptides interacted with the platelet integrin alpha(IIb)beta(3), but with greatly varying affinities. Our results suggest that the antiplatelet activity of BLAH(6) can be retained in albumin fusion proteins in which smaller peptides replace the barbourin domain; these proteins may be less immunogenic than BLAH(6).

  20. 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.

  1. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease

    PubMed Central

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K.; Gordeuk, Victor R.; Cho, Jaehyung

    2017-01-01

    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50–500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease. PMID:27758820

  2. ARQ 092, an orally-available, selective AKT inhibitor, attenuates neutrophil-platelet interactions in sickle cell disease.

    PubMed

    Kim, Kyungho; Li, Jing; Barazia, Andrew; Tseng, Alan; Youn, Seock-Won; Abbadessa, Giovanni; Yu, Yi; Schwartz, Brian; Andrews, Robert K; Gordeuk, Victor R; Cho, Jaehyung

    2017-02-01

    Previous studies identified the Ser/Thr protein kinase, AKT, as a therapeutic target in thrombo-inflammatory diseases. Here we report that specific inhibition of AKT with ARQ 092, an orally-available AKT inhibitor currently in phase Ib clinical trials as an anti-cancer drug, attenuates the adhesive function of neutrophils and platelets from sickle cell disease patients in vitro and cell-cell interactions in a mouse model of sickle cell disease. Studies using neutrophils and platelets isolated from sickle cell disease patients revealed that treatment with 50-500 nM ARQ 092 significantly blocks αMβ2 integrin function in neutrophils and reduces P-selectin exposure and glycoprotein Ib/IX/V-mediated agglutination in platelets. Treatment of isolated platelets and neutrophils with ARQ 092 inhibited heterotypic cell-cell aggregation under shear conditions. Intravital microscopic studies demonstrated that short-term oral administration of ARQ 092 or hydroxyurea, a major therapy for sickle cell disease, diminishes heterotypic cell-cell interactions in venules of sickle cell disease mice challenged with tumor necrosis factor-α. Co-administration of hydroxyurea and ARQ 092 further reduced the adhesive function of neutrophils in venules and neutrophil transmigration into alveoli, inhibited expression of E-selectin and intercellular adhesion molecule-1 in cremaster vessels, and improved survival in these mice. Ex vivo studies in sickle cell disease mice suggested that co-administration of hydroxyurea and ARQ 092 efficiently blocks neutrophil and platelet activation and that the beneficial effect of hydroxyurea results from nitric oxide production. Our results provide important evidence that ARQ 092 could be a novel drug for the prevention and treatment of acute vaso-occlusive complications in patients with sickle cell disease.

  3. An ultrastructural analysis of endothelial change paralleling platelet aggregation in a light/dye model of microvascular insult.

    PubMed Central

    Povlishock, J. T.; Rosenblum, W. I.; Sholley, M. M.; Wei, E. P.

    1983-01-01

    Those microvascular endothelial events that parallel the evolution of platelet aggregation were evaluated in a well-controlled animal model. Cat pial microvessels were observed through a cranial window while local platelet aggregation was produced by intravenous injection of sodium fluorescein and simultaneous exposure of the pial vessels to light from a filtered mercury lamp that excited the fluorescein. The vessels were fixed in situ when the in vivo observations of a preselected vessel indicated early, intermediate, or advanced aggregation in that vessel. The preselected vessel was then harvested for ultrastructural study together with adjacent vessels from the illuminated field. These vessels and appropriate controls were compared in semiserial thin sections. The onset of platelet aggregation in both venules and arterioles was accompanied by focal endothelial lucency, vacuole formation, luminal membrane rupture, and swelling of the nuclear envelope. These changes were not found in control material. With intermediate aggregation these changes were more common, while with advanced aggregation these abnormalities occurred together with focal endothelial denudation. Thus, in this model denudation occurred only with advanced aggregation and was not a prerequisite for aggregation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:6824062

  4. Aggregation and disaggregation kinetics of human blood platelets: Part I. Development and validation of a population balance method.

    PubMed Central

    Huang, P Y; Hellums, J D

    1993-01-01

    Hydrodynamic shear stress of sufficient intensity is known to cause platelet activation and aggregation and to alter the effects of biochemical platelet agonists and antagonists. In this work, a population balance equation (PBE) model is developed for analysis of platelet aggregation and disaggregation kinetics under the influence of a shear field. The model incorporates both aggregation and disaggregation by splitting and/or erosion mechanisms. This paper, the first of a series of three, deals with the formulation, simplification, and validation of the PBE and with the estimation of parameters involved in the PBE. These population parameters include collision efficiency, void fraction (related to the particle collision diameter), and the breakage rate coefficient. The platelet particle size distribution is determined experimentally, both initially and at some later times. The PBE can then be used to match satisfactorily the observed particle histograms, by appropriate choice of parameters of the model as functions of time, platelet size, and magnitude of physical or chemical stimuli. Besides providing information on adhesive forces and on the rates of aggregation and disaggregation, these parameters infer the physical properties of platelets and platelet aggregates. These properties are of potential value in increasing our understanding of the processes involved in thrombotic disease and/or therapy. A numerical procedure for solving the PBE is validated by application to simple cases for which analytical solutions are available. The model is applied to analysis of experiments, and parameter sensitivity studies are used to order the importance of the parameters and to reduce the complexity of the model. The simplified model is shown to give good agreement with experimental observations. PMID:8369441

  5. Investigation of platelet aggregation inhibitory activity by phenyl amides and esters of piperidinecarboxylic acids.

    PubMed

    de Candia, Modesto; Summo, Luciana; Carrieri, Antonio; Altomare, Cosimo; Nardecchia, Adele; Cellamare, Saverio; Carotti, Angelo

    2003-04-03

    A series of anilides and phenyl esters of piperidine-3-carboxylic acid (nipecotic acid) were synthesized and tested for the ability to inhibit aggregation of human platelet rich-plasma triggered by adenosine 5'-diphosphate (ADP) and adrenaline. As a rule, amides were about two times more active than the corresponding esters, and derivatives bearing substituents at the para position of the phenyl ring were significantly more active than the meta-substituted ones. Among the tested compounds, 4-hexyloxyanilide of nipecotic acid (18a) was found to be the most active one, its IC(50) value being close to that of the most active bis-3-carbamoylpiperidines reported in literature (ca. 40 micro M) and aspirin (ca. 60 microM) in ADP- and adrenaline-induced aggregation, respectively. Compared with the isomeric 4-hexyloxyanilides of piperidine-2-carboxylic (pipecolinic) and piperidine-4-carboxylic (isonipecotic) acids, compound 18a showed higher activity, and a Hansch-type quantitative structure-activity relationship (QSAR) study highlighted lipophilicity and increase in electron density of the phenyl ring as the properties which mainly increase the antiplatelet activity (r(2)=0.74, q(2)=0.64). The interaction of nipecotoyl anilides with phosphatidylinositol, a major component of the inner layer of the platelet membranes, was investigated by means of flexible docking calculation methods to give an account of a key event underlying their biological action.

  6. Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

    PubMed

    Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

    2003-05-01

    Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

  7. Purification and characterization of BmooAi: a new toxin from Bothrops moojeni snake venom that inhibits platelet aggregation.

    PubMed

    de Queiroz, Mayara Ribeiro; Mamede, Carla Cristine N; de Morais, Nadia Cristina G; Fonseca, Kelly Cortes; de Sousa, Bruna Barbosa; Migliorini, Thaís M; Pereira, Déborah Fernanda C; Stanziola, Leonilda; Calderon, Leonardo A; Simões-Silva, Rodrigo; Soares, Andreimar Martins; de Oliveira, Fábio

    2014-01-01

    In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

  8. Purification and Characterization of BmooAi: A New Toxin from Bothrops moojeni Snake Venom That Inhibits Platelet Aggregation

    PubMed Central

    Ribeiro de Queiroz, Mayara; Mamede, Carla Cristine N.; de Morais, Nadia Cristina G.; Cortes Fonseca, Kelly; Barbosa de Sousa, Bruna; Migliorini, Thaís M.; Pereira, Déborah Fernanda C.; Stanziola, Leonilda; Calderon, Leonardo A.; Simões-Silva, Rodrigo; Martins Soares, Andreimar; de Oliveira, Fábio

    2014-01-01

    In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders. PMID:24971359

  9. Acetylcholinesterase Inhibitors with Photoswitchable Inhibition of β-Amyloid Aggregation

    PubMed Central

    2014-01-01

    Photochromic cholinesterase inhibitors were obtained from cis-1,2-α-dithienylethene-based compounds by incorporating one or two aminopolymethylene tacrine groups. All target compounds are potent acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors in the nanomolar concentration range. Compound 11b bearing an octylene linker exhibited interactions with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Yet upon irradiation with light, the mechanism of interaction varied from one photochromic form to another, which was investigated by kinetic studies and proved “photoswitchable”. The AChE-induced β-amyloid (Aβ) aggregation assay gave further experimental support to this finding: Aβ1–40 aggregation catalyzed by the PAS of AChE might be inhibited by compound 11b in a concentration-dependent manner and seems to occur only with one photochromic form. Computational docking studies provided potential binding modes of the compound. Docking studies and molecular dynamics (MD) simulations for the ring-open and -closed form indicate a difference in binding. Although both forms can interact with the PAS, more stable interactions are observed for the ring-open form based upon stabilization of a water molecule network within the enzyme, whereas the ring-closed form lacks the required conformational flexibility for an analogous binding mode. The photoswitchable inhibitor identified might serve as valuable molecular tool to investigate the different biological properties of AChE as well as its role in pathogenesis of AD in in vitro assays. PMID:24628027

  10. The effect of physical exercise on the daily rhythm of platelet aggregation and body temperature in horses.

    PubMed

    Piccione, Giuseppe; Grasso, Fortunata; Fazio, Francesco; Giudice, Elisabetta

    2008-05-01

    The goal of this study was to investigate the influence of physical activity on the daily rhythm of platelet aggregation and body temperature in horses. Blood samples from 12 Thoroughbred horses, six sedentary animals and six athletes (studied both before and after a period of inactivity) were collected at 4h intervals for 48h via an intravenous cannula inserted into the jugular vein. Body temperature was recorded every 4h for 48h with a rectal probe. Platelet aggregation was measured with an aggregometer. Collagen was used to test the aggregation of the plasma samples. Statistical analysis of the data was performed by one-way repeated-measures analysis of variance (ANOVA) and by single cosinor method. Cosinor analysis identified the periodic parameters and their acrophases (expressed in hours) during the 2 days of monitoring. On each single day, there was a highly significant effect of time in all the horses, with P values <0.05. Temperature rhythms were unaffected by exercise. Platelet aggregation in exercising horses differed from the sedentary horses, and this difference disappeared after a 2-week period of rest. The results could be interpreted as indicating that physical exercise has an influence on the daily rhythm of platelet aggregation in horses.

  11. Retro-inverso peptide inhibitor nanoparticles as potent inhibitors of aggregation of the Alzheimer's Aβ peptide.

    PubMed

    Gregori, Maria; Taylor, Mark; Salvati, Elisa; Re, Francesca; Mancini, Simona; Balducci, Claudia; Forloni, Gianluigi; Zambelli, Vanessa; Sesana, Silvia; Michael, Maria; Michail, Christos; Tinker-Mill, Claire; Kolosov, Oleg; Sherer, Michael; Harris, Stephen; Fullwood, Nigel J; Masserini, Massimo; Allsop, David

    2017-02-01

    Aggregation of amyloid-β peptide (Aβ) is a key event in the pathogenesis of Alzheimer's disease (AD). We investigated the effects of nanoliposomes decorated with the retro-inverso peptide RI-OR2-TAT (Ac-rGffvlkGrrrrqrrkkrGy-NH2) on the aggregation and toxicity of Aβ. Remarkably low concentrations of these peptide inhibitor nanoparticles (PINPs) were required to inhibit the formation of Aβ oligomers and fibrils in vitro, with 50% inhibition occurring at a molar ratio of ~1:2000 of liposome-bound RI-OR2-TAT to Aβ. PINPs also bound to Aβ with high affinity (Kd=13.2-50 nM), rescued SHSY-5Y cells from the toxic effect of pre-aggregated Aβ, crossed an in vitro blood-brain barrier model (hCMEC/D3 cell monolayer), entered the brains of C57 BL/6 mice, and protected against memory loss in APPSWE transgenic mice in a novel object recognition test. As the most potent aggregation inhibitor that we have tested so far, we propose to develop PINPs as a potential disease-modifying treatment for AD.

  12. Venous levels of shear support neutrophil-platelet adhesion and neutrophil aggregation in blood via P-selectin and beta2-integrin

    NASA Technical Reports Server (NTRS)

    Konstantopoulos, K.; Neelamegham, S.; Burns, A. R.; Hentzen, E.; Kansas, G. S.; Snapp, K. R.; Berg, E. L.; Hellums, J. D.; Smith, C. W.; McIntire, L. V.; Simon, S. I.

    1998-01-01

    BACKGROUND: After activation, platelets adhere to neutrophils via P-selectin and beta2-integrin. The molecular mechanisms and adhesion events in whole blood exposed to venous levels of hydrodynamic shear in the absence of exogenous activation remain unknown. METHODS AND RESULTS: Whole blood was sheared at approximately 100 s(-1). The kinetics of neutrophil-platelet adhesion and neutrophil aggregation were measured in real time by flow cytometry. P-selectin was upregulated to the platelet surface in response to shear and was the primary factor mediating neutrophil-platelet adhesion. The extent of neutrophil aggregation increased linearly with platelet adhesion to neutrophils. Blocking either P-selectin, its glycoprotein ligand PSGL-1, or both simultaneously by preincubation with a monoclonal antibody resulted in equivalent inhibition of neutrophil-platelet adhesion (approximately 30%) and neutrophil aggregation (approximately 70%). The residual amount of neutrophil adhesion was blocked with anti-CD11b/CD18. Treatment of blood with prostacyclin analogue ZK36374, which raises cAMP levels in platelets, blocked P-selectin upregulation and neutrophil aggregation to baseline. Complete abrogation of platelet-neutrophil adhesion required both ZK36374 and anti-CD18. Electron microscopic observations of fixed blood specimens revealed that platelets augmented neutrophil aggregation both by forming bridges between neutrophils and through contact-mediated activation. CONCLUSIONS: The results are consistent with a model in which venous levels of shear support platelet adherence to neutrophils via P-selectin binding PSGL-1. This interaction alone is sufficient to mediate neutrophil aggregation. Abrogation of platelet adhesion and aggregation requires blocking Mac-1 in addition to PSGL-1 or P-selectin. The described mechanisms are likely of key importance in the pathogenesis and progression of thrombotic disorders that are exacerbated by leukocyte-platelet aggregation.

  13. Extract from Aronia melanocarpa fruits potentiates the inhibition of platelet aggregation in the presence of endothelial cells

    PubMed Central

    Luzak, Boguslawa; Golanski, Jacek; Rozalski, Marek; Krajewska, Urszula; Olas, Beata

    2010-01-01

    Introduction Some polyphenolic compounds extracted from Aronia melanocarpa fruits (AM) have been reported to be cardioprotective agents. In this study we evaluated the ability of AM extract to increase the efficacy of human umbilical vein endothelial cells (HUVECs) to inhibit platelet functions in vitro. Material and methods This study encompasses two models of monitoring platelet reactivity: optical aggregation and platelet degranulation (monitored as the surface CD62P expression) in PRP upon the stimulation with ADP. Results We observed that only at low concentrations (5 µg/ml) did AM extract significantly improve antiplatelet action of HUVECs towards ADP-activated platelets in the aggregation test. Conclusions It is concluded that the potentiating effect of AM extract on the endothelial cell-mediated inhibition of platelet aggregation clearly depends on the used concentrations of Aronia-derived active compounds. Therefore, despite these encouraging preliminary outcomes on the beneficial effects of AM extract polyphenols, more profound dose-effect studies should certainly be considered before the implementation of Aronia-originating compounds in antiplatelet therapy and the prevention of cardiovascular diseases. PMID:22371737

  14. First total synthesis of antrocamphin A and its analogs as anti-inflammatory and anti-platelet aggregation agents.

    PubMed

    Lee, Chia-Lin; Huang, Chi-Huan; Wang, Hui-Chun; Chuang, Da-Wei; Wu, Ming-Jung; Wang, Sheng-Yang; Hwang, Tsong-Long; Wu, Chin-Chung; Chen, Yeh-Long; Chang, Fang-Rong; Wu, Yang-Chang

    2011-01-07

    Naturally occurring antrocamphin A (1) is a potent anti-inflammatory compound from the edible fungus Antrodia camphorata (Taiwanofungus camphoratus), whose wild fruiting body is used as a valuable folk medicine in Taiwan. This study is the first total synthesis of antrocamphin A (1) and its analogs. Their inhibition ability on NO release, superoxide anion generation, elastase release and platelet aggregation are reported herein.

  15. Hemolysate-mediated platelet aggregation: an additional risk mechanism contributing to thrombosis of continuous flow ventricular assist devices.

    PubMed

    Tran, Phat L; Pietropaolo, Maria-Grazia; Valerio, Lorenzo; Brengle, William; Wong, Raymond K; Kazui, Toshinobu; Khalpey, Zain I; Redaelli, Alberto; Sheriff, Jawaad; Bluestein, Danny; Slepian, Marvin J

    2016-07-01

    Despite the clinical success and growth in the utilization of continuous flow ventricular assist devices (cfVADs) for the treatment of advanced heart failure, hemolysis and thrombosis remain major limitations. Inadequate and/or ineffective anticoagulation regimens, combined with high pump speed and non-physiological flow patterns, can result in hemolysis which often is accompanied by pump thrombosis. An unexpected increase in cfVADs thrombosis was reported by multiple major VAD implanting centers in 2014, highlighting the association of hemolysis and a rise in lactate dehydrogenase (LDH) presaging thrombotic events. It is well established that thrombotic complications arise from the abnormal shear stresses generated by cfVADs. What remains unknown is the link between cfVAD-associated hemolysis and pump thrombosis. Can hemolysis of red blood cells (RBCs) contribute to platelet aggregation, thereby, facilitating prothrombotic complications in cfVADs? Herein, we examine the effect of RBC-hemolysate and selected major constituents, i.e., lactate dehydrogenase (LDH) and plasma free hemoglobin (pHb) on platelet aggregation, utilizing electrical resistance aggregometry. Our hypothesis is that elements of RBCs, released as a result of shear-mediated hemolysis, will contribute to platelet aggregation. We show that RBC hemolysate and pHb, but not LDH, are direct contributors to platelet aggregation, posing an additional risk mechanism for cfVAD thrombosis.

  16. 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

  17. 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

  18. Flow-cytometric screening of aggregation-inhibitors using a fluorescence-assisted intracellular method.

    PubMed

    Lindberg, Hanna; Sandersjöö, Lisa; Meister, Sebastian W; Uhlén, Mathias; Löfblom, John; Ståhl, Stefan

    2017-01-01

    Aggregation of misfolded peptides and proteins is a key event in several neurodegenerative diseases. Suggested treatments of such disorders aim to inhibit the initial aggregation process. Here, we have developed an intracellular, function-based screening method, intended for isolation of aggregation-inhibitors from combinatorial protein libraries by flow-cytometric cell sorting. The method is based on fusion of aggregation-prone peptides to a fluorescent protein, functioning as a solubility reporter. Co-expression of a protein-based aggregation-inhibitor should prevent aggregation and thus increase the whole-cell fluorescence. We evaluated the method using the aggregation-prone Alzheimer's-related amyloid-β (Aβ) peptide in fusion to green-fluorescent protein (GFP), and an Aβ aggregation-inhibiting Affibody molecule. To adapt the method for library applications, the inhibitor was linked to an mCherry reporter for normalization of protein expression levels. We found that aggregation propensity correlates with fluorescence intensity, as co-expression of the Affibody-inhibitor increased the whole-cell fluorescence relative to a non-inhibitor. Employing improved cultivation parameters, we furthermore demonstrated efficient rescue from aggregation of an α-synuclein-derived protein using a different type of aggregation-inhibitor. Importantly, we also showed that the Aβ aggregation-inhibiting Affibody molecule could be isolated from a 1:10,000 background of non-inhibitors, with around 3,500-fold enrichment, in one cycle of fluorescence-based cell sorting. In conclusion, our new method represents a promising approach for generation of novel protein-based aggregation-inhibitors.

  19. Effects of the glycoprotein IIb/IIIa receptor antagonist c7E3 Fab and anticoagulants on platelet aggregation and thrombin potential under high coagulant challenge in vitro.

    PubMed

    Koestenberger, M; Gallistl, S; Cvirn, G; Roschitz, B; Muntean, W

    2000-07-01

    The present study was performed to investigate the combined effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab (abciximab) and the anticoagulants unfractionated heparin (UH), low molecular weight heparin (LMWH), and recombinant hirudin (rH) on platelet aggregation and thrombin generation under high coagulant challenge by extrinsic activation of platelet-rich plasma. Platelet aggregation and thrombin generation were assessed simultaneously in the presence of different concentrations of abciximab and anticoagulants. Increasing concentrations of abciximab resulted in a dose-dependent anti-aggregating effect with a maximum at 20 microg/ml. Doses of 5, 10, and 20 microg/ml abciximab prolonged the lag phase until the onset of platelet aggregation, but this effect was independent of the dosage used. Abciximab had no influence on the thrombin potential under our high coagulant challenge. UH, LMWH, and rH showed a dose-dependent prolongation of the lag phase until the onset of platelet aggregation and decreased the thrombin potential. Addition of anticoagulants did not contribute to further inhibition of platelet aggregation in the presence of abciximab, but the combination of abciximab and anticoagulants exhibited an additive effect on prolongation of the lag phase until the onset of platelet aggregation. Addition of abciximab to anticoagulants did not result in further decrease of the thrombin potential. Our study demonstrates the respective specific effects of abciximab and anticoagulants on platelet aggregation and thrombin potential under high coagulant challenge, and also an additive effect of abciximab and the anticoagulants UH, LMWH, and rH on the lag phase until the onset of platelet aggregation.

  20. The pyrrolidinoindoline alkaloid Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling

    PubMed Central

    Su, Xing-li; Su, Wen; Wang, Ying; Wang, Yue-hu; Ming, Xin; Kong, Yi

    2016-01-01

    Aim: Psm2, one of the pyrrolidinoindoline alkaloids isolated from whole Selaginella moellendorffii plants, has shown a potent antiplatelet activity. In this study, we further evaluated the antiplatelet effects of Psm2, and elucidated the underlying mechanisms. Methods: Human platelet aggregation in vitro and rat platelet aggregation ex vivo were investigated. Agonist-induced platelet aggregation was measured using a light transmission aggregometer. The antithrombotic effects of Psm2 were evaluated in arteriovenous shunt thrombosis model in rats. To elucidate the mechanisms underlying the antiplatelet activity of Psm2, ELISAs, Western blotting and molecular docking were performed. The bleeding risk of Psm2 administration was assessed in a mouse tail cutting model, and the cytotoxicity of Psm2 was measured with MTT assay in EA.hy926 cells. Results: Psm2 dose-dependently inhibited human platelet aggregation induced by ADP, U4619, thrombin and collagen with IC50 values of 0.64, 0.37, 0.35 and 0.87 mg/mL, respectively. Psm2 (1, 3, 10 mg/kg) administered to rats significantly inhibited platelet aggregation ex vivo induced by ADP. Psm2 (1, 3, 10 mg/mL, iv) administered to rats with the A–V shunt dose-dependently decreased the thrombus formation. Psm2 inhibited platelet adhesion to fibrinogen and collagen with IC50 values of 84.5 and 96.5 mg/mL, respectively, but did not affect the binding of fibrinogen to GPIIb/IIIa. Furthermore, Psm2 inhibited AktSer473 phosphorylation, but did not affect MAPK signaling and Src kinase activation. Molecular docking showed that Psm2 bound to phosphatidylinositol 3-kinase β (PI3Kβ) with a binding free energy of −13.265 kcal/mol. In addition, Psm2 did not cause toxicity in EA.hy926 cells and produced only slight bleeding in a mouse tail cutting model. Conclusion: Psm2 inhibits platelet aggregation and thrombus formation by affecting PI3K/Akt signaling. Psm2 may be a lead compound or drug candidate that could be developed for the

  1. Platelet adhesiveness and aggregation in congenital afibrinogenemia. An investigation of three patients with post-transfusion, cross-correction studies between two of them.

    PubMed

    Girolami, A; De Marco, L; Virgolini, L; Peruffo, R; Fabris, F

    1975-02-01

    Platelet adhesiveness and aggregation were studied in three patients with congenital afibrinogenemia. The results obtained may be summarized as follows: The retention of platelets to a glass-bead filter determined with the Salzman method was significantly decreased; it was normal after fibrinogen infusion. With a modification of the Hellem test the values obtained were slightly decreased. Adrenalin-induced aggregation was absent whereas ADP-and collagen-induced aggregation was near normal or slightly decreased. Thrombofax aggregation was absent in citrated plasma. The abnormalities of platelet aggregation were corrected after fibrinogen infusion or after addition in vitro of fibrinogen, hemofilia A plasma and PPP obtained from an afibrinogenemic patient after fibrinogen infusion. The abnormalities of platelet aggregation were corrected well by ADP, collagen and Thrombofax in heparinized blood, but only a slight correction of adrenalin-induced aggregation was noted. Thrombin aggregation proved to be normal with the higher concentrations, whereas it was defective with the lower ones. Ristocetin aggregation was normal in citrated plasma at the concentration of 1.5 mg per ml but it was absent at the lower concentration (1.0 mg per ml). Ristocetin aggregation was, on the other hand absent in heparinized blood regardless of the concentration. These findings are in agreement with the presence of a prolonged bleeding time in congenital afibrinogenemia and suggest that fibrinogen plays an important role in platelet aggregation and adhesiveness.

  2. Stoichiometry and Physical Chemistry of Promiscuous Aggregate-Based Inhibitors

    PubMed Central

    Coan, Kristin E. D.

    2009-01-01

    Many false positives in early drug discovery owe to nonspecific inhibition by colloid-like aggregates of organic molecules. Despite their prevalence, little is known about aggregate concentration, structure, or dynamic equilibrium; the binding mechanism, stoichiometry with, and affinity for enzymes remain uncertain. To investigate the elementary question of concentration, we counted aggregate particles using flow cytometry. For seven aggregate-forming molecules, aggregates were not observed until the concentration of monomer crossed a threshold, indicating a “critical aggregation concentration” (CAC). Above the CAC, aggregate count increased linearly with added organic material, while the particles dispersed when diluted below the CAC. The concentration of monomeric organic molecule is constant above the CAC, as is the size of the aggregate particles. For two compounds that form large aggregates, nicardipine and miconazole, we measured particle numbers directly by flow cytometry, determining that the aggregate concentration just above the CAC ranged from 5 to 30 fM. By correlating inhibition of an enzyme with aggregate count for these two drugs, we determined that the stoichiometry of binding is about 10 000 enzyme molecules per aggregate particle. Using measured volumes for nicardipine and miconazole aggregate particles (2.1 × 1011 and 4.7 × 1010 Å3, respectively), computed monomer volumes, and the observation that past the CAC all additional monomer forms aggregate particles, we find that aggregates are densely packed particles. Finally, given their size and enzyme stoichiometry, all sequestered enzyme can be comfortably accommodated on the surface of the aggregate. PMID:18588298

  3. A randomized, controlled study on the influence of acetaminophen, diclofenac, or naproxen on aspirin-induced inhibition of platelet aggregation.

    PubMed

    Galliard-Grigioni, Katja S; Reinhart, Walter H

    2009-05-01

    Nonsteroidal anti-inflammatory drugs (NSAID) may interfere with aspirin (acetylsalicylic acid) and increase the risk for cardiovascular events. The clinical relevance is uncertain. The aim of this study was to analyse the influence of a co-administration of aspirin and NSAID on platelet aggregation. In a randomized, placebo controlled trial, eleven healthy volunteers were studied during 4 separate study periods of 4 days each. Individuals were treated on each occasion with 100 mg aspirin daily in combination with either 3 x 1 g acetaminophen, 3 x 50 mg diclofenac, 3 x 250 mg naproxen, or 3 x 1 placebo. Primary hemostasis was assessed with a platelet function analyser (PFA-100), which measures the closure time (CT) of a collagen- and epinephrine-coated pore by aggregating platelets in flowing blood. Naproxen enhanced the anti-aggregatory action of aspirin after 24 h (CT rising from 104+/-16 s at baseline to 212+/-69 s at 24 h, P<0.001), which was not seen with any other drug combination. Diclofenac reduced the anti-aggregatory action of aspirin in the first two days, since the CT did not rise significantly (109+/-19 s, 148+/-56 s, and 168+/-66 s at 0 h, 24 h, 48 h, respectively, P>0.05). Acetaminophen had no effect compared with placebo. After 4 days of treatment platelet aggregation was similarly inhibited by all combinations. We conclude that a co-administration of NSAID and aspirin may interfere with platelet inhibition at the beginning of a treatment with an increase of naproxen and a decrease of diclofenac. This effect is lost after 4 days, suggesting that a regular daily co-administration of NSAID does not have an influence on platelet inhibition by aspirin.

  4. Rapid Purification and Procoagulant and Platelet Aggregating Activities of Rhombeobin: A Thrombin-Like/Gyroxin-Like Enzyme from Lachesis muta rhombeata Snake Venom

    PubMed Central

    Torres-Huaco, Frank Denis; Werneck, Cláudio C.; Vicente, Cristina Pontes; Vassequi-Silva, Talita; Nery-Diez, Ana Cláudia Coelho; Mendes, Camila B.; Antunes, Edson; Marangoni, Sérgio; Damico, Daniela C. S.

    2013-01-01

    We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BAρNA, with a broad optimum pH (7–10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, “ex vivo”, a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC). PMID:24058917

  5. Effects of calcium antagonists on serotonin-dependent aggregation and serotonin transport in platelets of patients with migraine.

    PubMed

    Pukhal'skaya, T G; Kolosova, O A; Men'shikov, M Y; Vein, A M

    2000-07-01

    Flunarizine and cinnarizine (IC50 6.8x10(-6) and 2.8x10(-5) M, respectively) inhibited 3H-serotonin uptake by platelets. In higher doses, they blocked serotonin-induced platelet aggregation and stimulated 3H-serotonin release from these cells. Imipramine did not affect serotonin-releasing effects of preparations. In all patients cinnarizine was more potent in inhibiting serotonin uptake, and in half of the patients cinnarizine displayed higher activity as an inductor of serotonin release.

  6. Prostaglandin E1-induced deconsolidation of thrombin-activated platelet aggregates I: ultrastructure-computer image analysis.

    PubMed

    Salganicoff, L; Russo, M A; Sevy, R W

    1999-06-15

    We have compared, at an ultrastructural-computer image morphometric level, the relaxation induced by Mg-ethylene-bis-oxyethylenenitrilo-tetracetic acid and prostaglandin E1 on a model of a thrombin-activated platelet aggregate. Mg-ethylene-bisoxyethylenenitrilo-tetracetic acid produced a small increase of 5.0% of the intercellular space over the control levels, and a decrease of 10.0+/-1.3% of the cross-sectional area of the platelets, with no apparent cytoskeletal alterations. In contrast, the prostaglandin El-treated preparation shows a 360% increase in the intercellular space and a decrease of the average platelet cross-sectional area of 30.0+/-2.0% with marked cytoskeletal alterations. We use the term "deconsolidation" to describe this effect. The enlargement of the intercellular space allows the observation of two types of contacts: (1) a type S (segmental) complex, of approximately 200-nm length that maintains a narrow interplatelet gap of 20-30 nm, filled with a dense intercellular material, and (2) a type R (reticular) complex, formed by scant focal regions of the plasma membrane from opposing platelets that are connected through a mesh of fibrillar or granular material contained within a variable-size space. We hypothesize that deconsolidation is caused by fluid loss from the platelets into the intercellular space. As a result, platelet volume decreases and intercellular space increases.

  7. Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction.

    PubMed

    Fisch, Adam S; Yerges-Armstrong, Laura M; Backman, Joshua D; Wang, Hong; Donnelly, Patrick; Ryan, Kathleen A; Parihar, Ankita; Pavlovich, Mary A; Mitchell, Braxton D; O'Connell, Jeffrey R; Herzog, William; Harman, Christopher R; Wren, Jonathan D; Lewis, Joshua P

    2015-01-01

    Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease.

  8. Genetic Variation in the Platelet Endothelial Aggregation Receptor 1 Gene Results in Endothelial Dysfunction

    PubMed Central

    Fisch, Adam S.; Yerges-Armstrong, Laura M.; Backman, Joshua D.; Wang, Hong; Donnelly, Patrick; Ryan, Kathleen A.; Parihar, Ankita; Pavlovich, Mary A.; Mitchell, Braxton D.; O’Connell, Jeffrey R.; Herzog, William; Harman, Christopher R.; Wren, Jonathan D.; Lewis, Joshua P.

    2015-01-01

    Platelet Endothelial Aggregation Receptor 1 (PEAR1) is a newly identified membrane protein reported to be involved in multiple vascular and thrombotic processes. While most studies to date have focused on the effects of this receptor in platelets, PEAR1 is located in multiple tissues including the endothelium, where it is most highly expressed. Our first objective was to evaluate the role of PEAR1 in endothelial function by examining flow-mediated dilation of the brachial artery in 641 participants from the Heredity and Phenotype Intervention Heart Study. Our second objective was to further define the impact of PEAR1 on cardiovascular disease computationally through meta-analysis of 75,000 microarrays, yielding insights regarding PEAR1 function, and predictions of phenotypes and diseases affected by PEAR1 dysregulation. Based on the results of this meta-analysis we examined whether genetic variation in PEAR1 influences endothelial function using an ex vivo assay of endothelial cell migration. We observed a significant association between rs12041331 and flow-mediated dilation in participants of the Heredity and Phenotype Intervention Heart Study (P = 0.02). Meta-analysis results revealed that PEAR1 expression is highly correlated with several genes (e.g. ANG2, ACVRL1, ENG) and phenotypes (e.g. endothelial cell migration, angiogenesis) that are integral to endothelial function. Functional validation of these results revealed that PEAR1 rs12041331 is significantly associated with endothelial migration (P = 0.04). Our results suggest for the first time that genetic variation of PEAR1 is a significant determinant of endothelial function through pathways implicated in cardiovascular disease. PMID:26406321

  9. Comparative evaluation of antiplatelet effect of lycopene with aspirin and the effect of their combination on platelet aggregation: An in vitro study

    PubMed Central

    Sawardekar, Swapna B.; Patel, Tejal C.; Uchil, Dinesh

    2016-01-01

    Introduction: The objective was to compare antiplatelet effect of lycopene with aspirin and to study effect of combination of the two on platelet aggregation in vitro, using platelets from healthy volunteers. Materials and Methods: Platelets were harvested; platelet count of platelet-rich plasma adjusted to 2.5 Χ 105/μL. Aspirin (140 μmol/L) and lycopene (4, 6, 8, 10, and 12 μmol/L) were studied in vitro against adenosine-5’- diphosphate (ADP) (2.5 μM/L) and collagen Results: All the concentrations of lycopene (4–12 μmol/L) exhibited reduction in maximum platelet aggregation induced by aggregating agents ADP and collagen (P < 0.01 vs. vehicle) and were comparable with aspirin. Lycopene at concentration 10 μmol/L showed maximum platelet inhibition (47.05% ± 19.56%) against ADP, whereas lycopene at concentration 8 μmol/L showed maximum platelet inhibition (54.26% ± 30.71%) against collagen. Four μmol/L of lycopene combined with 140 μmol/L and 70 μmol/L aspirin showed greater inhibition of platelets as compared to aspirin 140 μmol/L alone, against both ADP and collagen. Conclusion: The study favorably compares lycopene and aspirin with respect to their antiplatelet activities against ADP and collagen. Lycopene can be considered as a potential target for modifying the thrombotic and pro-inflammatory events associated with platelet activation. PMID:26997718

  10. Extracts from two frequently consumed spices--cumin (Cuminum cyminum) and turmeric (Curcuma longa)--inhibit platelet aggregation and alter eicosanoid biosynthesis in human blood platelets.

    PubMed

    Srivastava, K C

    1989-07-01

    In the traditional Indian system of medicine, Ayurveda, several spices and herbs are claimed to possess medicinal properties, such as being antithrombotic, antiatherosclerotic, hypolipidemic, anti-inflammatory etc. Earlier we have reported that extracts from several spices behave as antiaggregatory agents and inhibit eicosanoid synthesis. Similar studies with extracts prepared from cumin (Cuminum cyminum) and turmeric (Curcuma longa) were undertaken. Ethereal extract of both cumin and turmeric inhibited arachidonate-induced platelet aggregation. Extracts from these spices inhibited thromboxane B2 production from exogenous (14C) arachidonic acid (AA) in washed platelets; a simultaneous increase in the formation of lipoxygenase-derived products was observed. Less TxB2 was produced in blood samples treated with turmeric extract when they were allowed to clot. Turmeric extract inhibited incorporation of (14C)AA into platelet phospholipids and deacylation of AA-labelled phospholipids on stimulation with calcium ionophore A23187. Cumin extract was devoid of such effects. Extracts from the two spices reduced the formation of (14C)TxB2 from AA-labelled platelets when they were challenged with A23187. The anti-inflammatory property of turmeric may, in part, be explained by its effect on eicosanoid biosynthesis.

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  12. 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.

  13. Evaluation of antiplatelet effects of a modified protocol by platelet aggregation in patients undergoing "one-stop" hybrid coronary revascularization.

    PubMed

    Gao, Peixian; Xiong, Hui; Zheng, Zhe; Li, Lihuan; Gao, Runlin; Hu, Sheng-Shou

    2010-01-01

    "One-stop" hybrid coronary revascularization has emerged to be a reliable and attractive alternative for selected patients with multivessel coronary artery disease. However, the optimal antiplatelet regimen of the one-stop hybrid procedure still remains controversial. We modified the antiplatelet protocol in order to reduce the risk of perioperative bleeding and maximally inhibit platelet activity. This study sought to investigate whether the inhibition of platelet activity by this modified antiplatelet protocol is comparable with the conventional protocol widely used and recommended in percutaneous coronary interventions (PCI). Twenty three patients undergoing one-stop hybrid procedure and 20 patients undergoing conventional PCI were enrolled in this prospective study. The modified antiplatelet protocol included perioperative use of aspirin; clopidogrel was administered immediately before PCI with a 300 mg loading dose, followed by a maintenance dose of 75 mg/day for 12 months. Blood samples were obtained before the operation and 2 hours, day 1 and day 3 after operation. Platelet aggregation was induced with: 1) arachidonic acid (AA) (final concentration 0.5 mmol/L) to assess the efficacy of aspirin; 2) adenosine diphosphate (ADP) (final concentration 10 micromol/L) to assess the specific efficacy of clopidogrel. Platelet counts were statistically lower in the hybrid group than in the PCI control group (p = 0.0018) on day 1 after operation. AA-induced platelet aggregation increased significantly in comparison with the preoperative baseline values (p = 0.0079) and the PCI control group (p = 0.0023) on day 1 after operation. ADP-induced platelet aggregation gradually decreased in the hybrid group, and achieved similar platelet inhibition with the PCI group on 2 hours and day 1 after operation. No major adverse clinical events such as death, perioperative myocardial infarction, stent thrombosis or reoperation for bleeding occurred in both groups within 30 days after

  14. Aggregation of human platelets by endotoxic glycolipid-bearing Salmonella minnesota Re595 is prevented by synthetic peptide analogs of cell adhesion sites of fibrinogen and fibronectin

    SciTech Connect

    Timmons, S.; Grabarek, J.; Kloczewiak, M.; Hawiger, J.

    1986-03-01

    Thrombocytopenia often accompanies sepsis due to endotoxin producing gram-negative bacteria. The authors have observed that mutant Re595 of S. minnesota induced aggregation of human platelets separated from plasma fibrinogen (Theta) and other proteins. This aggregation is dependent on ADP secreted from storage granules in response to mutant Re595. Platelet aggregation induced by mutant Re595 was prevented by simultaneously added EDTA and EGTA (5mM), whereas secretion of /sup 14/C-serotonin was maintained. Preincubation of platelets with chelators (1 hr, 37/sup 0/C), known to dissociate irreversibly the platelet membrane glycoprotein IIb x IIIa complex, abolished aggregation while serotonin secretion was decreased by only one fourth. Since the GPIIb x IIIa complex constitutes the receptor for Theta, its role was examined using synthetic peptide analogs of sites on gamma and alpha chains of Theta. Gamma 400-411 (225 ..mu..M) inhibited platelet aggregation induced by mutant Re595 while serotonin secretion was unaffected. Alpha 572-575 (RGDS; 100 ..mu..M), analogous to cell adhesion site of fibronectin, also prevented aggregation induced by mutant Re595. Thus, mutant Re595 causes platelet aggregation which is divalent cation-dependent and proceeds via receptor pathway for secreted adhesive macromolecules.

  15. Metalloproteases Affecting Blood Coagulation, Fibrinolysis and Platelet Aggregation from Snake Venoms: Definition and Nomenclature of Interaction Sites

    PubMed Central

    Kini, R. Manjunatha; Koh, Cho Yeow

    2016-01-01

    Snake venom metalloproteases, in addition to their contribution to the digestion of the prey, affect various physiological functions by cleaving specific proteins. They exhibit their activities through activation of zymogens of coagulation factors, and precursors of integrins or receptors. Based on their structure–function relationships and mechanism of action, we have defined classification and nomenclature of functional sites of proteases. These metalloproteases are useful as research tools and in diagnosis and treatment of various thrombotic and hemostatic conditions. They also contribute to our understanding of molecular details in the activation of specific factors involved in coagulation, platelet aggregation and matrix biology. This review provides a ready reference for metalloproteases that interfere in blood coagulation, fibrinolysis and platelet aggregation. PMID:27690102

  16. Schisandra chinensis and Morus alba Synergistically Inhibit In Vivo Thrombus Formation and Platelet Aggregation by Impairing the Glycoprotein VI Pathway

    PubMed Central

    Sung, Yoon-Young; Kim, Seung Hyung; Park, Sun Haeng

    2017-01-01

    Morus alba L. (MAL) extract has been used in traditional medicine for its cardioprotective and antiplatelet effects, while another herbal remedy, Schisandra chinensis (SCC), has been reported to have anti-inflammatory and antioxidant properties. We evaluated underlying cellular changes exerted by extracts of these plants on platelet function and effects of SCC + MAL on in vivo thrombus formation using AV shunt and tail thrombosis-length models in rats. In vitro platelet aggregation, granule secretion, and [Ca2+]i release assays were carried out. The activation of integrin αIIbβ3 and phosphorylation of downstream signaling molecules, including MAPK and Akt, were investigated using cytometry and immunoblotting, respectively. Scanning electron microscopy (SEM) was used to evaluate changes in platelet shape and HPLC analysis was carried out to identify the marker compounds in SCC + MAL mixture. In vivo thrombus weight and average length of tail thrombosis were significantly decreased by SCC + MAL. In vitro platelet aggregation, granule secretion, [Ca2+]i release, and integrin αIIbβ3 activation were notably inhibited. SCC + MAL markedly reduced the phosphorylation of MAPK pathway factors along with Akt. HPLC analysis identified four marker compounds: isoquercitrin, astragalin, schizandrol A, and gomisin A. The extracts exerted remarkable synergistic effects as natural antithrombotic and antiplatelet agent and a potent drug candidate for treating cardiovascular diseases. PMID:28194217

  17. Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

    PubMed Central

    van Golen, Rowan F.; Stevens, Katarzyna M.; Colarusso, Pina; Jaeschke, Hartmut; Heger, Michal

    2016-01-01

    Background Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. Aims To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Methods Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Results Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Conclusions Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. Relevance for patients I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I

  18. Evening primrose oil ameliorates platelet aggregation and improves cardiac recovery in myocardial-infarct hypercholesterolemic rats

    PubMed Central

    Abo-Gresha, Noha M; Abel-Aziz, Eman Z; Greish, Sahar M

    2014-01-01

    Omega-6 polyunsaturated fatty acids (n-6 PUFA) are well known for their role in cardiovascular disease (CVD). We proposed that Evening prime rose oil (EPO) can improve the outcome of a heart with myocardial infarction (MI) in the presence of diet-induced hyperaggregability. This study was designed to examine its cholesterol lowering, antithrombotic and anti-inflammatory effects. High fat diet was administered for 4 weeks then MI was induced by isoproterenol (85 mg/kg/s.c./24 h). Treatment with EPO (5 or 10 gm/kg/day) for 6 weeks improved the electrocardiographic pattern, serum lipid profile, cardiac biomarkers as well as Platelet aggregation percent. We reported decreased serum level of TNF-α, IL-6 and COX-2 with attenuation of TNF-α and TGF-β in the cardiac homogenate. Moreover, histopathology revealed marked amelioration. Finally, we provide evidence that EPO improve cardiac recovery in hypercholesterolemic myocardial infarct rats. These effects are attributed to direct hypocholesterolemic effect and indirect effect on the synthesis of eicosanoids (prostaglandins, cytokines). PMID:24665356

  19. Effects of smoking on prostacyclin formation and platelet aggregation in users of oral contraceptives.

    PubMed

    Roy, S

    1999-06-01

    This review covers 10 articles published during 1981-96 to determine which subgroups within the population of smokers and oral contraceptive (OC) users are at especially elevated risk for thromboembolic events. It was found that the thrombogenic effects of smoking are mediated by two pathways: 1) increased biosynthesis of thromboxane, which promotes platelet aggregation; 2) degenerative changes in the vascular endothelium leading to the formation of atheromas and necrotic plaque, which in turn results in formation of thrombi. There was evidence showing that cardiovascular disease associated with OC use is due to thrombosis rather than atherosclerosis. The effects of smoking, resulting in an increase in arterial wall stiffness and changes in the patterns of arterial blood flow, would lead to formation of thrombi. However, there was an inconsistency in the data presented; studies revealed that smoking alone constitutes greater cardiovascular risk. Cigarette smoking and OC use can act synergistically to favor the formation of thrombi and increase the risk of thromboembolic events. Heavy smoking, a longer history of smoking, and inhaling smoke in association with OC use increases the risk of thrombosis.

  20. A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates

    PubMed Central

    Chen, Meimei; Ye, Xiaohui; Ming, Xin; Chen, Yahui; Wang, Ying; Su, Xingli; Su, Wen; Kong, Yi

    2015-01-01

    Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development. PMID:26035670

  1. Effect of GABA derivatives on the rate of thrombus formation, platelet aggregation, and plasma coagulation capacity in rats with experimental gestosis.

    PubMed

    Tyurenkov, I N; Perfilova, V N; Karamysheva, V I; Reznikova, L B; Mokrousov, I S; Mikhailova, L I; Berestovitskaya, V M; Vasil'eva, O S

    2014-12-01

    Experimental gestosis induced by replacement of drinking water with 1.8% NaCl promoted hypercoagulation, increased the rate and degree of platelet aggregation, and reduced clotting time in pregnant females. GABA derivatives, compounds RGPU-151, RGPU-152, and phenibut normalized parameters of hemostasis and platelet aggregation and the rate of thrombus formation in the animals. The efficiency of the test substances did not significantly differ from that of the reference drug sulodexide.

  2. Effect of a selective thromboxane synthase inhibitor on arterial graft patency and platelet deposition in dogs

    SciTech Connect

    McDaniel, M.D.; Huntsman, W.T.; Miett, T.O.; Cronenwett, J.L.

    1987-08-01

    This study examined the effect of selective thromboxane synthase inhibition and nonselective cyclooxygenase inhibition on vascular graft patency and indium 111-labeled platelet deposition in 35 mongrel dogs undergoing carotid artery replacement with 4 mm X 4 cm polytetrafluoroethylene (PTFE) (one side) and Dacron (opposite side) end-to-end grafts. Aspirin-dipyridamole therapy improved one-week graft patency, from 46% in untreated dogs to 93% in treated dogs. Thromboxane synthase inhibition (U-63557A) improved graft patency in these dogs to 81%. Both drug treatments reduced platelet deposition on Dacron and PTFE grafts by 48% to 68% compared with control dogs. Dacron grafts accumulated significantly more platelets than PTFE grafts but had comparable patency rates. Low-dose aspirin therapy had no significant effect on either graft patency or platelet deposition. All treatment groups showed a 60% to 76% reduction in serum thromboxane B2, but only thromboxane synthase inhibitor treatment increased plasma 6-keto-prostaglandin F1 alpha by 100%. Selective thromboxane synthase inhibition improved small-caliber prosthetic graft patency to the same extent as did conventional cyclooxygenase inhibition in this preliminary study.

  3. Heme Aggregation inhibitors: antimalarial drugs targeting an essential biomineralization process.

    PubMed

    Ziegler, J; Linck, R; Wright, D W

    2001-02-01

    Malaria, resulting from the parasites of the genus Plasmodium, places an untold burden on the global population. As recently as 40 years ago, only 10% of the world's population was at risk from malaria. Today, over 40% of the world's population is at risk. Due to increased parasite resistance to traditional drugs and vector resistance to insecticides, malaria is once again resurgent. An emergent theme from current strategies for the development of new antimalarials is that metal homeostasis within the parasite represents an important drug target. During the intra-erythrocytic phase of its life cycle, the malaria parasite can degrade up to 75% of an infected cell's hemoglobin. While hemoglobin proteolysis yields requisite amino acids, it also releases toxic free heme (Fe(III)PPIX). To balance the metabolic requirements for amino acids against the toxic effects of heme, malaria parasites have evolved a detoxification mechanism which involves the formation of a crystalline heme aggregate known as hemozoin. An overview of the biochemistry of the critical detoxification process will place it in the appropriate context with regards to drug targeting and design. Quinoline-ring antimalarial drugs are effective against the intraerythrocytic stages of pigment-producing parasites. Recent work on the mechanism of these compounds suggests that they prevent the formation of hemozoin. Evidence for such a mechanism is reviewed, especially in the context of the newly reported crystal structure of hemozoin. Additionally, novel drugs, such as the hydroxyxanthones, which have many of the characteristics of the quinolines are currently being investigated. Recent work has also highlighted two classes of inorganic complexes that have interesting antimalarial activity: (1) metal-N(4)O(2) Schiff base complexes and (2) porphyrins. The mechanism of action for these complexes is discussed. The use of these complexes as probes for the elucidation of structure-activity relationships in heme

  4. Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

    PubMed Central

    Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

    2014-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

  5. Attenuation of Thrombosis by Crude Rice (Oryza sativa) Bran Policosanol Extract: Ex Vivo Platelet Aggregation and Serum Levels of Arachidonic Acid Metabolites

    PubMed Central

    Ismail, Maznah; Tohit, Eusni Rahayu Mohd; Abdullah, Rasedee; Zhang, Yi-Da

    2016-01-01

    Background. Vascular occlusion or thrombosis was often attributed to uncontrolled platelet activation. Influence of sugarcane policosanol extract on platelet was reported but little was known of rice bran policosanol, particularly its mechanisms of actions on platelet activities. Objective. Antiplatelet mechanisms of rice bran policosanol extract (RBE) were studied using hyperlipidemic Sprague Dawley rats. Ex vivo platelet aggregation, platelet count (PC), bleeding time (BT), and coagulation time were assayed. Serum eicosanoids and other aggregation-related metabolites levels were quantified. Design. Rats were divided into 6 groups for comparisons (vehicle control Tween 20/H2O, high dose policosanol 500 mg/kg, middle dose policosanol 250 mg/kg, low dose policosanol 100 mg/kg, and positive control aspirin 30 mg/kg). Results. Low dose 100 mg/kg of RBE inhibited aggregation by 42.32 ± 4.31% and this was comparable with the effect of 30 mg/kg aspirin, 43.91 ± 5.27%. Results showed that there were no significant differences in PC, BT, and coagulation time among various groups after RBE treatment. Serum thromboxane A2 was attenuated while prostacyclin level increased upon RBE treatment. Conclusions. RBE reduced ex vivo ADP-induced platelet aggregation without giving adverse effects. No changes in full blood count suggested that rice bran policosanol did not disturb biological blood cell production and destruction yet it reduced aggregation through different mechanisms. PMID:27800004

  6. Activation of human platelets by antibodies to thymocytes and beta 2-microglobulin. I. Qualitative and quantitative aspects of the platelet aggregation induced by HATG and SA beta 2mG.

    PubMed Central

    Csákó, G; Suba, E A; Wistar, R

    1980-01-01

    The platelet effect of antibody preparations known to have immunosuppressive action was investigated by a turbidimetric method in vitro. Both horse anti-human thymocyte globulin (HATG) and sheep anti-human beta 2-microglobulin IgG (SA beta 2mG) caused the platelets to aggregate in all human platelet-rich plasmas (PRP) tested. The aggregation was usually irreversible and characterized by a sigmoid curve. The action is specific for the antibodies in HATG and SA beta 2mG because control preparations (horse normal IgG and IgG fractions of sheep normal, anti-dog IgG and anti-human IgA sera) were ineffective; further, heating (30 min at 56 degrees C) and BaSO4 or Al(OH)3 adsorption of HATG and SA beta 2mG did not alter their aggregating capability. When HATG and SA beta 2mG were added together to PRP, they induced aggregation in a simple additive manner. High antibody doses tended to decrease the extent of aggregation. The effect of platelet count on aggregation varied with both the dose level ('low' or 'high') and type (HATG or SA beta 2mG) of the inducer antibody. Using fixed submaximal doses, four main aggregation patterns could be recognized among 60 PRP: (i) high responses to both HATG and SA beta 2mG; (ii) high to HATG, low to SA beta 2mG; (iii) low to HATG, high to SA beta 2mG; and (iv) low to both. The results provide guidelines for quantitative aggregation studies with platelet antibodies and suggest that HATG and SA beta 2mG act through distinct platelet membrane components, the receptor for the latter being the best characterized protein of the mammalian cell membrane. PMID:6156042

  7. Platelet-Monocyte Aggregates and C-Reactive Protein are Associated with VTE in Older Surgical Patients

    PubMed Central

    Shih, Lauren; Kaplan, David; Kraiss, Larry W.; Casper, T. Charles; Pendleton, Robert C.; Peters, Christopher L.; Supiano, Mark A.; Zimmerman, Guy A.; Weyrich, Andrew S.; Rondina, Matthew T.

    2016-01-01

    Emerging evidence implicates platelets as key mediators of venous thromboembolism (VTE). Nevertheless, the pathways by which platelets and circulating procoagulant proteins synergistically orchestrate VTE remain incompletely understood. We prospectively determined whether activated platelets and systemic procoagulant factors were associated with VTE in 32 older orthopedic surgery patients. Circulating platelet-monocyte aggregates (PMAs), p-selectin expression (P-SEL), and integrin αIIbβ3 activation (PAC-1 binding) were assessed pre-operatively and 24 hours post-operatively. The proinflammatory and procoagulant molecule C-reactive protein (CRP), which induces PMA formation in vitro, along with plasma d-dimer and fibrinogen levels were also measured. The primary outcome was VTE occurring within 30 days post-operatively. Overall, 40.6% of patients developed VTE. Patients with VTE had a significant increase in circulating PMAs and CRP post-operatively, compared to those without VTE. Changes in PMA and CRP in VTE patients were significantly correlated (r2 = 0.536, p = 0.004). In contrast, P-SEL expression and PAC-1 binding, fibrinogen levels, and d-dimers were not associated with VTE. This is the first study to identify that increased circulating PMAs and CRP levels are early markers associated with post-surgical VTE. Our findings also provide new clinical evidence supporting the interplay between PMAs and CRP in patients with VTE. PMID:27270163

  8. Enhancement of platelet aggregation and thrombus formation by arsenic in drinking water: a contributing factor to cardiovascular disease.

    PubMed

    Lee, Moo-Yeol; Bae, Ok-Nam; Chung, Seung-Min; Kang, Kyu-Tae; Lee, Joo-Young; Chung, Jin-Ho

    2002-03-01

    Arsenic in drinking water is a worldwide health problem that is associated with cardiovascular disease, but the cause is currently unknown. Arsenic effects on platelets, which are important in development of cardiovascular disease, were examined in vitro and in a drinking water study using a rat animal model. Trivalent inorganic arsenic (arsenite) induced in vitro aggregation when platelets were exposed to subthreshold challenge by thrombin and several other agonists in a concentration-dependent manner, with arsenite being the most potent form tested. Arsenite also induced significant increases in serotonin secretion, thromboxane A(2) formation, and adhesion protein expression in platelets. Consistent with the in vitro studies, 4-week ingestion of arsenite-contaminated drinking water resulted in enhanced arterial thrombosis. Human platelets showed similar responses, suggesting that the effects seen in animal experiments are applicable to humans. These results will provide new insights into the mechanism of arsenic-induced cardiovascular disease. They will also allow regulatory agencies to estimate risk from arsenic-induced cardiovascular disease and to determine if drinking water regulatory levels based on human cancer studies will protect against noncancer effects associated with cardiovascular disease.

  9. Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia

    PubMed Central

    Major, Heather D.; Cambell, Robert A.; Silver, Robert M.; Branch, D. Ware; Weyrich, Andrew S.

    2014-01-01

    Objective Soluble fms-like tyrosine kinase (sFlt-1) is an important mediator in the pathogenesis of preeclampsia. We sought to determine if platelet-monocyte aggregates (PMAs) produced sFlt-1 and if PMAs contributed to sFlt-1 production in preeclampsia. Study Design Case-control study of sFlt-1 release from PMAs using blood samples from women with preeclampsia matched by gestational age to pregnant controls. A third group of nonpregnant, reproductive-age women comprised an additional control group. Experiments were also performed using blood from non-pregnant women to elucidate if inducing PMAs could stimulate sFlt-1 production, and if so, to determine the necessary receptors and pathways. Results Women with preeclampsia had increased total Flt-1 concentrations in platelets and monocytes at baseline compared to pregnant controls (25 vs. 10 pg/ml, p=0.0003). sFlt-1 production was elicited from monocytes incubated with thrombin-activated platelets from non-pregnant women. sFlt-1 production was regulated at the transcriptional level by p38 and NF-κB dependent pathways. Conclusion Activated platelets in preeclampsia bind monocytes to generate sFlt-1. PMAs are a previously unrecognized source of sFlt-1 that may contribute to endothelial dysfunction and systemic inflammation commonly observed in preeclampsia. PMID:24440566

  10. Purification and characterization of L-amino acid oxidase from king cobra (Ophiophagus hannah) venom and its effects on human platelet aggregation.

    PubMed

    Li, Z Y; Yu, T F; Lian, E C

    1994-11-01

    Venoms of several snake species contain large amounts of L-amino acid oxidase but its effects on human plasma coagulation and platelet aggregation have not been explored. We have purified L-amino acid oxidase from king cobra venom through CM-Sephadex C-25, Sephadex G-100 and DEAE Sephadex A-50 chromatographies. The purified enzyme has a mol. wt of 135,000 as determined by gel filtration and 65,000 by SDS-PAGE under non-reducing and reducing conditions. Incubation of plasma with L-amino acid oxidase at 200 micrograms/ml did not affect prothrombin time, activated partial thromboplastin time, or thrombin time. Upon addition of L-amino acid oxidase, platelets in platelet-rich plasma were aggregated. The enzyme-induced aggregation was abolished by catalase. The aggregation was also inhibited by indomethacin, aspirin, ethylenediaminetetraacetate, sodium nitroprusside, prostaglandin E1, mepacrine and verapamil, but not by heparin, hirudin, creatine phosphate/creatine phosphokinase or antimycin/2-deoxy-D-glucose. These results suggest that L-amino acid oxidase induces human platelet aggregation through the formation of H2O2, and subsequent thromboxane A2 synthesis requiring Ca2+ but independent of ADP release. The platelet aggregation caused by L-amino acid oxidase is likely to contribute to toxicity inflicted by cobra venom.

  11. Relation between clopidogrel active metabolite levels and different platelet aggregation methods in patients receiving clopidogrel and aspirin.

    PubMed

    Liang, Yan; Johnston, Marilyn; Hirsh, Jack; Pare, Guillaume; Li, Chunjian; Mehta, Shamir; Teo, Koon K; Sloane, Debi; Yi, Qilong; Zhu, Jun; Eikelboom, John W

    2012-11-01

    Clopidogrel is a prodrug that undergoes bioconversion via cytochrome P450 system to form an active metabolite (AM) that binds to the platelet ADP receptor. The antiplatelet effect of clopidogrel is commonly assessed by measuring the aggregatory response to 5 μM ADP by light transmission aggregation (LTA) or multiple electrode aggregometry (MEA) or by the vasodilator-stimulated phosphoprotein platelet reactivity index (VASP-PRI). To determine which of these three tests of platelet ADP receptor pathway inhibition most closely correlates with clopidogrel AM levels. We analyzed blood samples from 82 patients with coronary artery disease who were randomized to receive double-dose or standard dose clopidogrel for 2 weeks. We measured peak clopidogrel AM levels, platelet aggregation in response to ADP and VASP-PRI on days 1, and repeated all the measures on days 7 and 14. Linear regression analysis was used to examine the correlation between clopidogrel AM and LTA, MEA and VASP-PRI. Bland-Altman plots were used to explore the agreement between tests of the antiplatelet effects of clopidogrel. Clopidogrel AM on day 1 correlated most closely with VASP-PRI (r = -0.5767) and demonstrated weaker correlations with LTA (r = -0.4656) and MEA (r = -0.3384) (all p < 0.01). Intra-class correlation (ICC) between VASP-PRI and LTA was 0.6446; VASP-PRI and MEA was 0.4720; and LTA and MEA was 0.4693. Similar results were obtained on days 7 and 14. Commonly used pharmacodynamic measures of clopidogrel response are only moderately correlated with clopidogrel AM levels and may not be suitable to measure the adequacy of clopidogrel therapy.

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

    PubMed Central

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

    2014-01-01

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

  13. Abciximab treatment in vitro after aspirin treatment in vivo has additive effects on platelet aggregation, ATP release, and P-selectin expression.

    PubMed

    Scazziota, A; Altman, R; Rouvier, J; Gonzalez, C; Ahmed, Z; Jeske, W P; Walenga, J M; Fareed, J

    2000-12-15

    To prevent arterial thrombosis, abciximab is administered together with aspirin. However, whether or not there are benefits to combine abciximab with aspirin is not yet well defined. Healthy volunteers were studied for the effect of aspirin + abciximab using sodium arachidonate and adenosine diphosphate (ADP) alone or in combination to induce platelet activation/aggregation. Abciximab produced complete inhibition of platelet aggregation induced with ADP but only 40% inhibition of aggregation induced by 0.75-mmol/l sodium arachidonate. Abciximab added in vitro to platelet-rich plasma (PRP) from platelets from aspirin-treated donors produced an almost complete inhibition of platelet aggregation. Aspirin, and abciximab alone, did not inhibit adenosine triphosphate (ATP) release as thoroughly as aspirin + abciximab did. Abciximab (3-5 microg/ml) produced inhibition of P-selectin expression induced with 5 (from 46.2 +/- 6.0% to 27.4 +/- 7.0%, P=0.002) and 20-micromol/l ADP (from 53.1 +/- 8.1% to 35.1 +/- 11.0%, P=0.019), but no effect was observed when 0.75-mmol/l sodium arachidonate was used (P=0.721). Aspirin diminished P-selectin expression in sodium arachidonate-stimulated platelets (from 77.7 +/- 11.8% to 40.2 +/- 3.6%, P<0.0001) in non-aspirinated and platelets from aspirin-treated donors, respectively. Abciximab (3, 4, and 5 microg/ml) added to platelets from aspirin-treated donors decreased P-selectin expression in platelets stimulated with sodium arachidonate from 40.2 +/- 8.6% to 25.6 +/- 11.5% (P=0.027), to 20.5 +/- 3.5% (P<0.0001), and to 22.5 +/- 1.8% (P<0.0001). We concluded that the antiplatelet effect of abciximab is greatly increased by aspirin.

  14. Effects of ticlopidine or ticlopidine plus aspirin on platelet aggregation and ATP release in normal volunteers: why aspirin improves ticlopidine antiplatelet activity.

    PubMed

    Altman, R; Scazziota, A; Rouvier, J; Gonzalez, C

    1999-10-01

    Aspirin and ticlopidine are used to prevent arterial thrombosis. In some clinical settings ticlopidine is administered with aspirin for improving antithrombotic effect. We administered aspirin (100 mg/day), ticlopidine (500 mg/day), or ticlopidine and aspirin for 7 days to healthy volunteers. Platelet aggregation and ATP release induced by sodium arachidonate, ADP, or a combination of both were measured. Sodium arachidonate (0.25 mmol/L), which produces no platelet aggregation, combined with adenosine diphosphate (1 mumol/L), which produced a reversible platelet aggregation of 20% after ticlopidine, resulted in a synergistic platelet aggregation response in normal (74.6 +/- 9.2%) and in ticlopidine platelet-rich plasma (59.1% +/- 14.9%, p < 0.0001). Synergism after sodium arachidonate (0.75 mmol/L) plus adenosine diphosphate (4 mumol/L) fell from 75.8% +/- 11.0% and 59.1% +/- 15.6% after ticlopidine or aspirin, respectively, to 14.8% +/- 18.0% (p < 0.0001) after ticlopidine plus aspirin. Aspirin and ticlopidine alone did not inhibit adenosine triphosphate release as thoroughly as did aspirin plus ticlopidine. Aspirin or ticlopidine does not adequately prevent platelet activity as ticlopidine plus aspirin do. Addition of aspirin to treatment with ticlopidine improves their antiplatelet activity and better results could be obtained in arterial thrombotic prevention strategies.

  15. Alterations in the extracellular catabolism of nucleotides and platelet aggregation induced by high-fat diet in rats: effects of α-tocopherol.

    PubMed

    Gutierres, Jessié M; Carvalho, Fabiano B; Schetinger, Maria Rosa C; Rodrigues, Marília V; Vieira, Juliano M; Maldonado, Paula; Araújo, Maria do Carmo S; Schmatz, Roberta; Stefanello, Naiara; Jaques, Jeandre A S; Costa, Marcio; Morsch, Vera; Mazzanti, Cinthia M; Pimentel, Victor; Lopes, Sonia Terezinha A; Spanevello, Roselia M

    2014-06-01

    The aim of this study was to assess whether α-tocopherol administration prevented alterations in the ectonucleotidase activities and platelet aggregation induced by high-fat diet in rats. Thus, we examined four groups of male rats which received standard diet, high-fat diet (HFD), α-tocopherol (α-Toc), and high-fat diet plus α-tocopherol. HFD was administered ad libitum and α-Toc by gavage using a dose of 50 mg/kg. After 3 months of treatment, animals were submitted to euthanasia, and blood samples were collected for biochemical assays. Results demonstrate that NTPDase, ectonucleotide pyrophosphatase/phosphodiesterase, and 5'-nucleotidase activities were significantly decreased in platelets of HFD group, while that adenosine deaminase (ADA) activity was significantly increased in this group in comparison to the other groups (P < 0.05). When rats that received HFD were treated with α-Toc, the activities of these enzymes were similar to the control, but ADA activity was significantly increased in relation to the control and α-Toc group (P < 0.05). HFD group showed an increased in platelet aggregation in comparison to the other groups, and treatment with α-Toc significantly reduced platelet aggregation in this group. These findings demonstrated that HFD alters platelet aggregation and purinergic signaling in the platelets and that treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.

  16. Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase

    PubMed Central

    Sherrill-Mix, Scott; Hwang, Young; Eilers, Grant; McDanal, Charlene; Wang, Ping; Temelkoff, David

    2016-01-01

    The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization. PMID:27935939

  17. Isolation of microtubule coils from platelets after exposure to aggregating agents.

    PubMed Central

    White, J. G.; Krumwiede, M.; Burris, S. M.; Heagan, B.

    1986-01-01

    The discoid shape of human blood platelets is supported by a circumferential microtubule (MT) organized in many loops or coils. A recent study reported from the authors' laboratory demonstrated that significant numbers of MT rings could be isolated from resting platelets by simultaneous exposure to detergent and a small amount of fixative. This method has been used in the present investigation to determine the number of MT coils obtained from platelets after activation by ADP, thrombin, and the calcium ionophore, A23187. Concentrations of the agonists that caused shape change and internal transformation in parallel samples did not influence the frequency of MT rings present in activated samples after treatment with fixative and detergent. As many or more MT coils were present 5, 15, 30, 60, 90, and 120 seconds after addition of an agonist as from the control. Statistical analysis revealed no significant difference between the number of isolated coils from controls and activated platelets at any time during early activation. Immunofluorescence microscopic examination of platelets stained with a monoclonal antibody to tubulin at intervals of 5, 15, 30, 60, 90, and 120 seconds after activation on glass surfaces confirmed the suggestion that platelet MTs are resistant to disassembly during the early response to stimulation. Images Figure 1 Figure 7 and 8 Figure 9-14 PMID:3098108

  18. Mechanism of platelet activation induced by endocannabinoids in blood and plasma.

    PubMed

    Brantl, S Annette; Khandoga, Anna L; Siess, Wolfgang

    2014-01-01

    Platelets play a central role in atherosclerosis and atherothrombosis, and circulating endocannabinoids might modulate platelet function. Previous studies concerning effects of anandamide (N-arachidonylethanolamide) and 2-arachidonoylglycerol (2-AG) on platelets, mainly performed on isolated cells, provided conflicting results. We therefore investigated the action of three main endocannabinoids [anandamide, 2-AG and virodhamine (arachidonoylethanolamine)] on human platelets in blood and platelet-rich plasma (PRP). 2-AG and virodhamine induced platelet aggregation in blood, and shape change, aggregation and adenosine triphosphate (ATP) secretion in PRP. The EC50 of 2-AG and virodhamine for platelet aggregation in blood was 97 and 160 µM, respectively. Lower concentrations of 2-AG (20 µM) and virodhamine (50 µM) synergistically induced aggregation with other platelet stimuli. Platelet activation induced by 2-AG and virodhamine resembled arachidonic acid (AA)-induced aggregation: shape change, the first platelet response, ATP secretion and aggregation induced by 2-AG and virodhamine were all blocked by acetylsalicylic acid (ASA) or the specific thromboxane A2 (TXA2) antagonist daltroban. In addition, platelet activation induced by 2-AG and virodhamine in blood and PRP were inhibited by JZL184, a selective inhibitor of monoacylglycerol lipase (MAGL). In contrast to 2-AG and virodhamine, anandamide, a substrate of fatty acid amidohydrolase, was inactive. Synthetic cannabinoid receptor subtype 1 (CB1) and 2 (CB2) agonists lacked stimulatory as well as inhibitory platelet activity. We conclude that 2-AG and virodhamine stimulate platelets in blood and PRP by a MAGL-triggered mechanism leading to free AA and its metabolism by platelet cyclooxygenase-1/thromboxane synthase to TXA2. CB1, CB2 or non-CB1/CB2 receptors are not involved. Our results imply that ASA and MAGL inhibitors will protect platelets from activation by high endocannabinoid levels, and that

  19. High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors.

    PubMed

    Pujols, Jordi; Peña-Díaz, Samuel; Conde-Giménez, María; Pinheiro, Francisca; Navarro, Susanna; Sancho, Javier; Ventura, Salvador

    2017-03-02

    An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson's disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds.

  20. High-Throughput Screening Methodology to Identify Alpha-Synuclein Aggregation Inhibitors

    PubMed Central

    Pujols, Jordi; Peña-Díaz, Samuel; Conde-Giménez, María; Pinheiro, Francisca; Navarro, Susanna; Sancho, Javier; Ventura, Salvador

    2017-01-01

    An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds. PMID:28257086

  1. Computational selection of inhibitors of Abeta aggregation and neuronal toxicity.

    PubMed

    Chen, Deliang; Martin, Zane S; Soto, Claudio; Schein, Catherine H

    2009-07-15

    Alzheimer's disease (AD) is characterized by the cerebral accumulation of misfolded and aggregated amyloid-beta protein (Abeta). Disease symptoms can be alleviated, in vitro and in vivo, by 'beta-sheet breaker' pentapeptides that reduce plaque load. However the peptide nature of these compounds, made them biologically unstable and unable to penetrate membranes with high efficiency. The main goal of this study was to use computational methods to identify small molecule mimetics with better drug-like properties. For this purpose, the docked conformations of the active peptides were used to identify compounds with similar activities. A series of related beta-sheet breaker peptides were docked to solid state NMR structures of a fibrillar form of Abeta. The lowest energy conformations of the active peptides were used to design three dimensional (3D)-pharmacophores, suitable for screening the NCI database with Unity. Small molecular weight compounds with physicochemical features and a conformation similar to the active peptides were selected, ranked by docking and biochemical parameters. Of 16 diverse compounds selected for experimental screening, 2 prevented and reversed Abeta aggregation at 2-3microM concentration, as measured by Thioflavin T (ThT) fluorescence and ELISA assays. They also prevented the toxic effects of aggregated Abeta on neuroblastoma cells. Their low molecular weight and aqueous solubility makes them promising lead compounds for treating AD.

  2. SIRT1 prevents pulmonary thrombus formation induced by arachidonic acid via downregulation of PAF receptor expression in platelets.

    PubMed

    Kim, Yun Hak; Bae, Jin Ung; Kim, In Suk; Chang, Chulhun L; Oh, Sae Ock; Kim, Chi Dae

    2016-12-01

    SIRT1, a class III histone deacetylase, is critically involved in cellular response to stress and modulates cardiovascular risk factors. However, its role in thrombus formation is largely unknown. Thus, this study investigated the effect of SIRT1 on pulmonary thrombus formation, and then identified its role in the modulation of platelet aggregation. In isolated human platelets, cell aggregation was increased by various platelet activators, such as platelet activating factor (PAF), arachidonic acid (AA), ADP, and thrombin. AA- and PAF-mediated platelet aggregations were suppressed by WEB2086, a PAF receptor (PAFR) antagonist. Pulmonary thrombus formation induced by PAF or AA was also attenuated by WEB2086, suggesting that PAFR plays a key role in AA-induced platelet aggregation. In platelets isolated from SIRT1-TG mice as well as in platelets treated with resveratrol or reSIRT1, PAFR expression was decreased, whereas this expressional downregulation by SIRT1 activators was inhibited in platelets treated with MG132 (a proteasome inhibitor) or NH4Cl (a lysosome inhibitor). Furthermore, platelet aggregation induced by AA was markedly attenuated by resveratrol and reSIRT1. Likewise, the increased pulmonary thrombus formation in mice treated with AA was also attenuated by SIRT1 activators. In line with these results, pulmonary thrombus formation was markedly attenuated in SIRT1-TG mice. Taken together, this study showed that SIRT1 downregulates PAFR expression on platelets via proteasomal and lysosomal pathways, and that this downregulation inhibits platelet aggregation in vitro and pulmonary thrombus formation in vivo.

  3. 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.

  4. Experimental Trypanosoma cruzi cardiomyopathy in BALB/c mice. The potential role of intravascular platelet aggregation in its genesis.

    PubMed Central

    Rossi, M. A.; Gonçalves, S.; Ribeiro-dos-Santos, R.

    1984-01-01

    In male BALB/c mice aged 5-6 weeks inoculated three times at intervals of 15 days with 1 X 10(7) epimastigote forms of the PF strain of Trypanosoma cruzi and challenged 30 days after the last inoculation with 2 X 10(4) trypomastigote forms of the Colombia strain of T cruzi (the mice were sacrificed 80-100 days after the challenge) a cardiomyopathy very similar to that observed in the chronic phase of Chagas' disease in man develops. The cardiac syndrome is characterized grossly by cardiomegaly with hypertrophy, dilatation of ventricular chambers, and thinning of the apex of the left ventricle (apical aneurysm) and microscopically by focal areas of myocytolytic necrosis and myocardial degeneration with an inflammatory response composed of mononuclear cells (predominantly macrophages and a few lymphocytes) with concurrent interstitial fibrosis and occasional myofibers containing pseudocysts. In addition, aggregated platelets and occlusive thrombi were found in small epicardial and intramyocardial vessels of infected mice as compared with controls. The potential role of intravascular platelet aggregation in the causation of focal myocardial necrosis and degeneration and apical aneurysm in experimental T cruzi cardiomyopathy in BALB/c mice is discussed. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 PMID:6230012

  5. A Novel Pentapeptide Targeting Integrin β3-Subunit Inhibits Platelet Aggregation and Its Application in Rat for Thrombosis Prevention

    PubMed Central

    Qu, Qingrong; Liu, Yamin; Yan, Xuejiao; Fan, Xiaobo; Liu, Naifeng; Wu, Guoqiu

    2016-01-01

    Background: Antiplatelet therapy plays a pivotal role in the prevention and treatment of thrombotic diseases. We reported the screening of P1C as a novel integrin-binding peptide from the C-terminal of connective tissue growth factor. Primary study indicated that P1C has potential against platelet aggregation. Objectives: We aimed to find the shortest active unit from the P1C fragments and explore its in vivo and in vitro activities. Methods: A series of truncated P1C fragments was prepared and screened for antiplatelet activity. The most active fragment was evaluated using coagulation assays. Flow cytometry and confocal microscopy were used to determine the interaction between the peptide and the integrin. The in vivo potential was further explored using two types of rat models. Results: From a series of truncated P1C forms, a so-called P1Cm peptide of 5-amino acids, namely, IRTPK was screened out as the shortest active unit with superior activity. Coagulation experiments and an in vivo toxicity assay demonstrated that P1Cm is safe in vivo and inhibits ADP- and TH-induced human platelet aggregation in vitro in a concentration-dependent manner. Furthermore, it has limited effect on the coagulation parameters. Flow cytometry and confocal microscopy experiments consistently indicated that the peptide specifically binds the β3-subunit of integrin on platelets. Further experiments using rat models of artery-vein shunt and carotid arterial thrombosis illustrated that P1Cm can effectively prevent thrombosis formation. Conclusion: P1Cm may be a new, promising antithrombotic alternative to currently available antiplatelet treatments. PMID:27014063

  6. Hemostatic markers and platelet aggregation factors as predictive markers for type of stroke and neurological disability following cerebral infarction.

    PubMed

    Tombul, Temel; Atbas, Cigdem; Anlar, Omer

    2005-05-01

    We investigated the plasma levels of D-dimer, fibrinogen, beta-thromboglobulin (BTG) and platelet factor-4 (PF-4), indices of the occurrence of platelet activation in vivo, to find out their role in pathophysiology of ischemic stroke and whether or not such a role has any effect on the disability and the prognosis of stroke patients. A total of 76 patients with AIS aged from 26 to 85 (32 men, 44 women) and 30 cases as controls with similar age (18 men, 12 women) were included in the study. The plasma levels of D-dimer, BTG and PF-4 were measured by ELISA method using a special commercial kit. The cases were allocated into two groups as non-embolic (NEI) and cardioembolic stroke (CEI). The D-dimer levels in 76% of 42 patients in NEI group (p<0.05) and 85.2% of 34 patients in CEI group (p<0.05) were outside the confidence interval (CI) defined for the control group. The levels of BTG were elevated in 81% of 42 cases with NEI (p<0.05) and in 76% of 34 cases with CEI, with reference to CI of control group. The levels of PF-4 were significantly increased in 86% of cases with NEI (p<0.05) and in 88% of cases with CEI than controls (p<0.05). It was observed that the cases with high Rankin scores had higher levels of D-dimer (p<0.005), BTG (p<0.01) and PF-4 (p<0.01) than those with lower scores. There was a correlation between hemostatic markers, platelet activation and functional disability. D-dimer levels were an important marker that determined to degree of the activation of hemostatic system, especially in CEI subtype. The platelet aggregation had an important role in pathophysiology of ischemic stroke and this condition is significant in NEI subgroup and subjects with large infarcts and high disability scores.

  7. [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.

  8. Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease.

    PubMed

    Harrington, Charles R; Storey, John M D; Clunas, Scott; Harrington, Kathleen A; Horsley, David; Ishaq, Ahtsham; Kemp, Steven J; Larch, Christopher P; Marshall, Colin; Nicoll, Sarah L; Rickard, Janet E; Simpson, Michael; Sinclair, James P; Storey, Lynda J; Wischik, Claude M

    2015-04-24

    Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μM. The Ki value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μM. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μM) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μM) required to reverse behavioral deficits and pathology in Tau transgenic mice.

  9. Structure and oxidation capacity of amino acid chloramine derivatives and their effects on platelet aggregation.

    PubMed

    Murina, M A; Chudina, N A; Roshchupkin, D I; Belakina, N S; Sergienko, V I

    2004-12-01

    Comparison of antiaggregation capacity of N-chloramine acids with different position of the chloramine group in the molecule showed that in the most efficient compounds the distance between the chloramine and carboxyl groups was 3-5 carbon atoms. This feature of antiaggregation activity was not related to the difference in oxidation capacity of N-chloramine acids. It was hypothesized that the revealed structural dependence of antiaggregation activity of N-chloramine acids is determined by the structure of platelet membrane, in particular, the presence of a negatively charged group near the site of interaction between N-chloramine acids and platelet membrane.

  10. [The relationship between lipid peroxidation and platelet aggregation in atherosclerotic patients].

    PubMed

    Gómez Calviño, C; Simón Carballo, R; Coma Alfonso, C; Sánchez de León, T; Montero Pacheco, E; Rodríguez Piloto, R

    1991-01-01

    We studied 58 patients with arterial esteno-occlusive disease, 32 diabetics and 26 nondiabetics. Some parameters of lipid metabolism and platelet function were evaluated. We show the correlations founded among these parameters and we offer a possible explanation which support this behaviour.

  11. Heparin-induced platelet aggregation (H-IPA): dose/response relationship for two low molecular weight (LMW) heparin preparations (CY 216 and CY 222)

    SciTech Connect

    Brace, L.D.; Fareed, J.

    1986-03-01

    The authors have previously demonstrated that heparin and a LMW heparin derivative (PK 10169) causes platelet aggregation in a dose-dependent manner that can be inhibited by antagonists of the thromboxane pathway. Using fractions of these agents separated on the basis of molecular weight (MW) by gel permeation chromatography, the authors showed that H-IPA was directly dependent upon the MW of the agents tested. In order to further examine this MW dependence, the authors tested two other LMW heparin preparations, CY 216 and CY 222 and subfractions of these agents separated on the basis of MW. Citrate anticoagulated whole blood was drawn from drug-free normal healthy donors whose platelets aggregated when heparin was added to their platelet-rich plasma (PRP). PRP was prepared, various concentrations of the agents or their subfractions were added and aggregation was monitored for 40 minutes at 37/sup 0/C. The results demonstrate that like heparin and PK 10169, CY 216 and CY 222 caused platelet aggregation in a dose and MW dependent manner. Fractions with MW less than 2500 daltons caused aggregation only at concentrations exceeding the therapeutic range of the agents. The authors conclude that the ability to cause H-IPA is an inherent property of heparin and its fractions.

  12. Monitoring of Intracellular Tau Aggregation Regulated by OGA/OGT Inhibitors.

    PubMed

    Lim, Sungsu; Haque, Md Mamunul; Nam, Ghilsoo; Ryoo, Nayeon; Rhim, Hyewhon; Kim, Yun Kyung

    2015-08-26

    Abnormal phosphorylation of tau has been considered as a key pathogenic mechanism inducing tau aggregation in multiple neurodegenerative disorders, collectively called tauopathies. Recent evidence showed that tau phosphorylation sites are protected with O-linked β-N-acetylglucosamine (O-GlcNAc) in normal brain. In pathological condition, tau is de-glycosylated and becomes a substrate for kinases. Despite the importance of O-GlcNAcylation in tau pathology, O-GlcNAc transferase (OGT), and an enzyme catalyzing O-GlcNAc to tau, has not been carefully investigated in the context of tau aggregation. Here, we investigated intracellular tau aggregation regulated by BZX2, an inhibitor of OGT. Upon the inhibition of OGT, tau phosphorylation increased 2.0-fold at Ser199 and 1.5-fold at Ser396, resulting in increased tau aggregation. Moreover, the BZX2 induced tau aggregation was efficiently reduced by the treatment of Thiamet G, an inhibitor of O-GlcNAcase (OGA). Our results demonstrated the protective role of OGT in tau aggregation and also suggest the counter-regulatory mechanism of OGA and OGT in tau pathology.

  13. The effect of different doses and different routes of acetylsalicylic acid administration on platelet aggregation in healthy volunteers and ischemic stroke patients.

    PubMed

    Chýlová, Miroslava; Moťovská, Zuzana; Osmančík, Pavel; Procházka, Bohumír; Kalvach, Pavel

    2015-04-01

    The purpose was to assess the effect of different doses and different routes of acetylsalicylic acid (ASA) administration on platelet aggregation and the comparison between platelet aggregation after the single and the repetitive administration of ASA in healthy individuals and in patients after ischemic stroke. The study group consists of 22 healthy individuals and 30 patients with documented ischemic stroke. Platelet aggregation was measured in healthy individuals: (a) twice before ASA and (b) 2 h after different single doses and different routes of ASA administration-(b1) 500 mg orally, (b2) 500 mg intravenously, and (b3) 100 mg orally. We measured aggregability in healthy individuals after five consecutive days of 100 mg of ASA q.d. and in patients on chronic ASA 100 mg q.d. The VerifyNow was used with results expressed in aspirin reaction units (ARU). In healthy individuals, the dose-(b1) 500 mg orally-reduced the aggregability to mean (SD) 392 (36) ARU (p < 0.001), (b2) 500 mg intravenously to 428 (46) (p < 0.001) and (b3) 100 mg orally to 460 (76) (p < 0.001). The suppression of aggregation after 500 mg was (p = 0.029) higher after the oral compared to intravenous administration, and the same is true for the suppression after single dose of 500 mg orally and 100 mg orally (p = 0.011). Oral dose 100 mg for 5 days in healthy individuals reduced aggregation to 405 (37) and in post-stroke patients to 433 (54). All doses of ASA, both orally and intravenously, have produced a significant reduction of platelet aggregation. Preference of the parenteral to oral application has not been established.

  14. Circulating complexes in leprosy studied by the platelet aggregation test. The platelet aggregation test and its relation to the rubino test and other sero-immunological parameters in 135 patients with leprosy

    PubMed Central

    Wager, O.; Penttinen, K.; Almeida, J. D.; Opromolla, D. V. A.; Godal, T.; Kronvall, G.

    1978-01-01

    Sera from 135 patients with leprosy were tested by the platelet aggregation test (PAT), by the Rubino test and by other sero-immunological assays. PAT positivity (titre≥10) was 53% in the lepromatous subgroups and 5% in the tuberculoid subgroups (P<0·005). The higher PAT titres and Rubino titres clustered significantly (P<0·0005) toward the lepromatous end of the disease spectrum. A statistically significant correlation was found between the PAT and the Rubino titres (0·05>P>0·025). Removal of the effect of the disease spectrum, however, resulted in a partial correlation between the PAT and the Rubino titres that was not significant (P>0·1), suggesting different basic mechanisms for the platelet aggregation (PA) and the Rubino activity of the lepromatous sera. The correlation between the PAT titres and twenty-nine other sero-immunological parameters was calculated, and a highly significant correlation was found between the PAT and the IgG level (P<0·005) and between the PAT and the antistaphylolysin-α titre (P<0·005). The PA activity in most lepromatous sera studied sedimented in the heavy (>19S) fractions and was inhibitable by IgM rheumatoid factor. It thus fulfilled the criteria for IgG complexes as defined in previous studies with known model Ag/Ab complexes and with sera from patients with immune complex states. The addition of an excess of soluble mycobacterial antigens affected the PA activity of some lepromatous sera, which suggests that the putative complexes were composed of mycobacterial antigens complexed with corresponding IgG antibody. It was concluded that the PAT is a sensitive detector of IgG complexes peculiar to the lepromatous leprosy. In leprosy the discriminatory power of the PAT seems to be superior to that of other immune complex tests recently applied for the analysis of leprosy series. ImagesFIG. 1 PMID:369750

  15. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: II. Inhibitor library, screening, and experimental validation.

    PubMed

    Zhang, Lin; Zhang, Chao; Sun, Yan

    2014-04-29

    Platelet adhesion on collagen mediated by integrin α2β1 has been proven important in arterial thrombus formation, leading to an exigent demand on development of potent inhibitors for the integrin α2β1-collagen binding. In the present study, a biomimetic design strategy of platelet adhesion inhibitors was established, based on the affinity binding model of integrin proposed in part I. First, a heptapeptide library containing 8000 candidates was designed to functionally mimic the binding motif of integrin α2β1. Then, each heptapeptide in the library was docked onto a collagen molecule for the assessment of its affinity, followed by a screening based on its structure similarity to the original structure in the affinity binding model. Eight candidates were then selected for further screening by molecular dynamics (MD) simulations. Thereafter, three candidates chosen from MD simulations were separately added into the physiological saline containing separated integrin and collagen, to check their abilities for blocking the integrin-collagen interaction using MD simulations. Of these three candidates, significant inhibition was observed in the presence of LWWNSYY. Finally, the binding affinity of LWWNSYY for collagen was demonstrated by isothermal titration calorimetry. Moreover, significant inhibition of platelet adhesion in the presence of LWWNSYY has been experimentally validated. This work has thus developed an effective strategy for the biomimetic design of peptide-based platelet adhesion inhibitors.

  16. 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

  17. Combined administration of 5-HT2 and thromboxane A2 antagonists: effects on platelet aggregation and isolated cardiac muscle

    PubMed Central

    Shaw, Linda A; Batey, Andrew J; Coker, Susan J

    1997-01-01

    To investigate possible mechanisms underlying the ability of combined administration of a 5-hydroxytryptamine2 (5-HT2) antagonist and a thromboxane A2 antagonist to reduce reperfusion-induced arrhythmias, the effects of these drugs alone and in combination on platelet aggregation and on cardiac muscle were determined. Platelet aggregation was measured in whole blood obtained from anaesthetized rats. Concentrations of 5-HT (10 μM) and the thromboxane A2 mimetic U46619 (1 μM) which did not cause aggregation themselves, enhanced the responses to ADP (0.1 μM) and to collagen (1 μg ml−1). For example, the response of 1.0±0.5 Ω to ADP alone was increased significantly to 6.4±1.0 Ω by 5-HT, 15.5±2.8 Ω by U46619, and 17.3±1.3 Ω when U46619, 5-HT and ADP were added together. In further experiments blood was obtained from rats which had received either the 5-HT2 antagonist, ICI 170,809 (1 mg kg−1), or the thromboxane A2 antagonist, ICI 192,605 (1 mg kg−1 min−1), or both in combination. When ADP was used as the primary aggregating agent, the ability of U46619 alone, or together with 5-HT, to enhance responses was reduced significantly by ICI 192,605 alone and in combination with ICI 170,809. Similar results were obtained with lower doses of ICI 170,809 (0.3 mg kg−1) and ICI 192,605 (0.3 mg kg−1 min−1). When collagen was used as the primary aggregating agent ICI 170,809 (1 mg kg−1) reduced the response to 5-HT (5.0±0.8 Ω versus 10.9±1.2 Ω in controls), and ICI 192,605 (1 mg kg−1 min−1) reduced the response to U46619 (6.8±2.5 Ω versus 11.2±2.2 Ω in control). The greatest reduction of platelet aggregation was seen in blood from rats which had received both antagonists, with the response to U46619 plus 5-HT plus collagen being 2.7± 0.6 Ω compared to 14.2±1.7 Ω in controls. In contrast, there was no significant attenuation of platelet aggregation in blood from rats which had received the lower

  18. Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

    PubMed Central

    Camargo, L.M.; França, C.N.; Izar, M.C.; Bianco, H.T.; Lins, L.S.; Barbosa, S.P.; Pinheiro, L.F.; Fonseca, F.A.H.

    2014-01-01

    It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1platelet aggregation, the amount of circulating endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation. PMID:24760119

  19. The effect of a prostaglandin DP-receptor partial agonist (192C86) on platelet aggregation and the cardiovascular system in healthy volunteers.

    PubMed

    Gray, S J; Giles, H; Posner, J

    1992-10-01

    1. PGD2 (DP)-receptors mediate inhibition of platelet aggregation and vasodilatation. If receptor reserve were greater on platelets it might be possible to separate these effects. To determine whether such a difference in receptor reserve exists, we have examined the effects of a highly selective DP-receptor partial agonist 192C86 on platelet aggregation and the cardiovascular system in healthy volunteers. 2. Using an open, dose-escalating study design, four male volunteers received constant rate intravenous infusions of 192C86 for up to 60 min. Ex vivo platelet aggregation to ADP and collagen in platelet-rich plasma (PRP) and whole blood (WB) was studied at baseline, after 15, 30 and 60 min of each infusion and at 180 min post-infusion. Heart rate (HR), systolic and diastolic (DBP) blood pressure were measured at frequent intervals. Adverse experiences were monitored by checklist. Facial flushing was assessed by the volunteer using a visual analogue scale, by an observer using a numerical scale and by full-face colour photographs. Blood was taken for assay of plasma 192C86 concentrations by radio-immunoassay (r.i.a.). 3. 192C86 (0.007-0.058 micrograms kg-1 min-1) inhibited platelet aggregation to ADP and collagen both in PRP and WB in a dose-dependent manner. However, this was always accompanied by a decrease in DBP, increase in HR and facial flushing. Plasma concentrations of 192C86 were at or below the limits of sensitivity of the r.i.a. (0.5 ng ml-1). 4. The highest infusion rate was stopped after 20 min due to symptomatic hypotension on standing.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. The effects of two synthetic glycoprotein IIb/IIIa antagonists, Ro 43-8857 and L-700,462, on platelet aggregation and bleeding in guinea-pigs and dogs: evidence that Ro 43-8857 is orally active.

    PubMed

    Cook, N S; Bruttger, O; Pally, C; Hagenbach, A

    1993-11-15

    In vitro platelet aggregation studies in whole blood were used to define the species-specificity profile of two synthetic GP-IIb/IIIa antagonists, Ro 43-8857 and L-700,462. Aggregation of rhesus monkey platelets was inhibited with a similar potency to human platelets, whereas both compounds were poor antagonists in mini-pig, rabbit or hamster blood. Compared to human platelets, Ro 43-8857 was 2-3-fold less active as an inhibitor of dog and guinea-pig platelet aggregation, whereas L-700,462 was, respectively, 4- and 14-fold less active in these species. In vivo investigations with these two compounds were performed in anesthetized guinea-pigs and conscious dogs, with bleeding times measured on small mesenteric arteries or on the inner jowl respectively. Ex vivo ADP-induced whole blood platelet aggregation was completely inhibited in guinea-pigs by Ro 43-8857 following intravenous administration of 0.1 mg/kg and intraduodenal administration of 3 mg/kg, with a duration of action exceeding 5 hours. Mesenteric bleeding times were prolonged by Ro 43-8857 only at doses causing supra-maximal inhibition of aggregation, suggesting these two effects could be partially dissociated. L-700,462 (3 mg/kg i.v.) was shorter acting than Ro 43-8857 in guinea-pigs (duration approximately 1 hour) and the anti-aggregatory effect was accompanied by mesenteric bleeding time prolongations. In conscious dogs, ex vivo aggregation was inhibited to approximately 80% by Ro 43-8857 (0.3 mg/kg i.v. or 10 mg/kg p.o.) and L-700,462 (1 mg/kg i.v.). However, bleeding time prolongations accompanied these anti-aggregatory effects with both compounds. In conclusion, we have shown clear differences between two synthetic GP-IIb/IIIa antagonists, both in terms of their species-specificity in vitro and in terms of their in vivo profile, and in particular the propensity to promote bleeding from mesenteric arteries in guinea-pigs. However, the ability of Ro 43-8857 to discriminate between anti-aggregatory and

  1. A novel family of RGD-containing disintegrin (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets integrins αIIbβ3 and αVβ3 and inhibits platelet aggregation and angiogenesis

    PubMed Central

    Ma, Dongying; Xu, Xueqing; An, Su; Liu, Huan; Yang, Xuening; Andersen, John F.; Wang, Yipeng; Tokumasu, Fuyuki; Ribeiro, José M. C.; Francischetti, Ivo M. B.; Lai, Ren

    2012-01-01

    A novel family of RGD-containing molecule (Tablysin-15) has been molecularly characterized from the salivary gland of the hematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity for platelet αIIbβ3 and endothelial cell αvβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~ 1 nM) and marginally to fibronectin (IC50 ~ 1 µM), but not to collagen. It also inhibits FGF-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilized fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-15. Moreover, immobilized Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow, without affecting platelet adhesion to collagen fibrils. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors. PMID:21475772

  2. 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.

  3. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  4. Morphologies developed by the drying of droplets containing dispersed and aggregated layered double hydroxide platelets.

    PubMed

    Zhang, Yan; Evans, Julian R G

    2013-04-01

    Despite much interest in the structures formed from droplets of suspension as they dry, there are few studies involving plate-like particles. Layered double hydroxide suspensions were prepared with pH adjusted to give well-dispersed and flocculated variants that were characterised by sedimentation and rheology measurements. In the well-dispersed suspension, the three-phase boundary was pinned and radial flow created a peripheral wall. The platelet structure involved local flat packing but was replete with scrolls that result from the recirculation flows in the droplets as they dry. In contrast, the flocculating suspension produced flatter droplet relics and the microstructure consisted of ordered domains which were disoriented with respect to each other. Although of scientific interest, the control of these structures will make it possible to use direct ink-jet printing to build 3D shapes for the preparation of aligned, ordered nanocomposites based on plate-like particles in which platelet preferred orientation mimics the structures found in nacre.

  5. Effects of LY117018 and the estrogen analogue, 17alpha-ethinylestradiol, on vascular reactivity, platelet aggregation, and lipid metabolism in the insulin-resistant JCR:LA-cp male rat: role of nitric oxide.

    PubMed

    Russell, J C; McKendrick, J D; Dubé, P J; Dolphin, P J; Radomski, M W

    2001-01-01

    The JCR:LA-cp rat is obese and insulin resistant and develops a major vasculopathy, with associated ischemic damage to the heart. Male rats were treated with 17alpha-ethinylestradiol (EE), LY117018, and/or the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME). LY117018 decreased plasma cholesterol esters, with a 40% reduction in total cholesterol. EE increased triglyceride levels and modestly decreased cholesterol esters. L-NAME increased blood pressure and aortic contractile sensitivity to phenylephrine and inhibited acetylcholine-induced relaxation. LY117018 decreased the force of contraction. The L-NAME-mediated increase in force of contraction and decrease in response to acetylcholine was inhibited by LY117018. L-NAME-induced hypertension was prevented by LY117018. Platelet aggregation was not different between obese and lean rats and was unaffected by L-NAME. LY117018, both in the absence and presence of L-NAME, inhibited platelet aggregation. The effects of LY117018 are apparently mediated through both NO-dependent and -independent mechanisms. The changes induced by EE and LY117018 may reflect the activation of multiple mechanisms, both estrogen receptor-dependent and -independent. The changes induced by LY117018 are significant and may prove to be cardioprotective in the presence of the insulin resistance syndrome.

  6. Aggregation of dipolar molecules in SiO2 hybrid organic-inorganic films: use of silver nanoparticles as inhibitors of molecular aggregation

    NASA Astrophysics Data System (ADS)

    Franco, Alfredo; García-Macedo, Jorge; Brusatin, Giovanna; Guglielmi, Massimo

    2013-04-01

    The technological implementation of hybrid organic-inorganic materials in second order nonlinear optical photonic devices depends strongly on the ability of the host matrixes to contain high loads of dipolar molecules without aggregation. Some organic molecules are often used to diminish the attracting interactions between dipolar molecules in such kind of materials, but their efficiency as inhibitors of molecular aggregation is limited by their polarizability. In this work, we report the use of silver nanoparticles as inhibitors of molecular aggregation in hybrid organic-inorganic films doped with dipolar molecules. The large polarizability of the silver nanoparticles makes them ideal moieties for the inhibition of the electrostatic interactions between dipolar nonlinear optical molecules. The average size of the silver nanoparticles in this work was 70.5 nm in diameter, they were synthesized using silver nitrate (AgNO3) as precursor and aminoethylaminopropyltrimethoxysilane as reducing agent. These nanoparticles were immersed in SiO2 hybrid organic-inorganic sol-gel films doped with dipolar chromophores to study their effect as inhibitors of dipolar chromophores aggregation. The presence of the silver nanoparticles in the solid films was confirmed by transmission electronic microscopy and UV-Visible spectroscopy. UV-Visible spectroscopy was also used to monitor the dipolar chromophores aggregation in the SiO2 films. We found that, at room temperature, silver nanoparticles are good inhibiting chromophores aggregation in comparison with the performance of organic inhibitors.

  7. Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness*

    PubMed Central

    Lalo, Ulyana; Jones, Sarah; Roberts, Jonathan A.; Mahaut-Smith, Martyn P.; Evans, Richard J.

    2012-01-01

    We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies. PMID:22851178

  8. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

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

    PubMed

    Lee, Ye-Ming; Hsieh, Kuo-Hsien; Lu, Wan-Jung; Chou, Hsiu-Chu; Chou, Duen-Suey; Lien, Li-Ming; Sheu, Joen-Rong; Lin, Kuan-Hung

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca(2+)](i) mobilization, thromboxane A(2) formation, hydroxyl radical (OH(●)) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A(2) formation, thereby leading to inhibition of [Ca(2+)](i) and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

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

    PubMed Central

    Lee, Ye-Ming; Hsieh, Kuo-Hsien; Lu, Wan-Jung; Chou, Hsiu-Chu; Chou, Duen-Suey; Lien, Li-Ming; Sheu, Joen-Rong; Lin, Kuan-Hung

    2012-01-01

    Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH●) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases. PMID:22611436

  11. Cangrelor attenuates coated-platelet formation.

    PubMed

    Norgard, Nicholas B; Hann, Callie L; Dale, George L

    2009-01-01

    P2Y(12) inhibitors were introduced clinically as effective inhibitors of adenosine-5'-diphosphate (ADP) mediated platelet activation and aggregation. This class of pharmacological agents has enjoyed considerable success. Cangrelor is a recently developed P2Y(12) inhibitor that has the advantage of being an active drug not requiring metabolic conversion, although it is not orally available. Coated-platelets are a subclass of activated platelets generated on dual agonist activation with collagen plus thrombin; the primary hallmark of coated-platelets is their ability to support prothrombinase activity. Interestingly, we recently observed that the relatively weak agonist ADP potentiates the production of coated-platelets by the very strong agonists collagen plus thrombin, a previously unknown role for ADP. The authors sought in this study to determine if P2Y(12) inhibitors, such as cangrelor, were capable of attenuating this augmentation of coated-platelet generation. Cangrelor, at physiologically relevant concentrations, was able to eliminate the ADP-dependent increase in coated-platelet production with an IC(50) of 1.4 nM. Cangrelor, however, had no effect on thrombin-dependent platelet activation as measured by P-selectin expression. Although this in vitro study does not address the question of whether the effectiveness of cangrelor in vivo is partially due to an attenuation of coated-platelet production in addition to its documented antiaggregatory effects, it does reveal an unexpected action of cangrelor. Additional studies will be required to determine if all P2Y(12) inhibitors are equally effective in attenuating coated-platelet production.

  12. 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.

  13. Variance of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) concentrations in activated, concentrated platelets from healthy male donors

    PubMed Central

    2014-01-01

    Background The use of autologous blood concentrates, such as activated, concentrated platelets, in orthopaedic clinical applications has had mixed results. Research on this topic has focused on growth factors and cytokines, with little directed towards matrix metalloproteinases (MMPs) which are involved in post-wound tissue remodeling. Methods In this study, the authors measured the levels of MMP-2, MMP-9 and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), in activated platelets derived from blood of healthy, male volunteers (n = 92), 19 to 60 years old. The levels of the natural inhibitors of these proteases, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2 and TIMP-4 were also assessed. Results Notably, there was no significant change in concentration with age in four of six targets tested. However, TIMP-2 and TIMP-4 demonstrated a statistically significant increase in concentration for subjects older than 30 years of age compared to those 30 years and younger (P = 0.04 and P = 0.04, respectively). Conclusion TIMP-2 and TIMP-4 are global inhibitors of MMPs, including MMP-2 (Gelatinase A). MMP-2 targets native collagens, gelatin and elastin to remodel the extracellular matrix during wound healing. A decreased availability of pharmacologically active MMP-2 may diminish the effectiveness of the use of activated, concentrated platelets from older patients, and may also contribute to longer healing times in this population. PMID:24766991

  14. Role of oxidative stress on platelet hyperreactivity during aging.

    PubMed

    Fuentes, Eduardo; Palomo, Iván

    2016-03-01

    Thrombotic events are common causes of morbidity and mortality in the elderly. Age-accelerated vascular injury is commonly considered to result from increased oxidative stress. There is abundant evidence that oxidative stress regulate several components of thrombotic processes, including platelet activation. Thus oxidative stress can trigger platelet hyperreactivity by decreasing nitric oxide bioavailability. Therefore oxidative stress measurement may help in the early identification of asymptomatic subjects at risk of thrombosis. In addition, oxidative stress inhibitors and platelet-derived nitric oxide may represent a novel anti-aggregation/-activation approach. In this article the relative contribution of oxidative stress and platelet activation in aging is explored.

  15. Encapsulation of eptifibatide in RGD-modified nanoliposomes improves platelet aggregation inhibitory activity.

    PubMed

    Bardania, Hassan; Shojaosadati, Seyed Abbas; Kobarfard, Farzad; Dorkoosh, Farid; Zadeh, Marjan Esfahani; Naraki, Mahmoud; Faizi, Mehrdad

    2017-02-01

    Eptifibatide is an antiplatelet drug used for the treatment of thrombosis. However, as a result of its accumulation in non-targeted tissues and short half-life, it has a limited efficacy. In this study, RGD-modified nano-liposomes (RGD-MNL) were prepared as carriers for the targeted delivery of eptifibatide to activated platelets. The nano-liposomes were about 90 ± 10 nm in size, with an encapsulation efficiency of 37 ± 5 % and a good stability during 21 days, with a negligible change in the size of nanoliosomes. The in vitro cytotoxicity of nanoliposomes was examined using MTT assay. The results obtained from the ex vivo study showed that the antiplatelet activity of eptifibatide encapsulated nanoliposomes was higher in comparison with the free drug (81.63 vs. 46.17 % for RGD-MNL) and (66.67 vs. 46.17 % for UNL), and this increase was more significant for nanoliposomes targeted with RGD peptide (81.63 %; p < 0.05). The results indicated that RGD-MNL encapsulated eptifibatide had no significant cytotoxic effect on cells. In conclusion, the present nanoliposome formulation can be regarded as a new delivery system for protection and enhancement of the antiplatelet activity of eptifibatide.

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

    PubMed Central

    Puy, Cristina; Tucker, Erik I.; Ivanov, Ivan S.; Gailani, David; Smith, Stephanie A.; Morrissey, James H.; Gruber, András; McCarty, Owen J. T.

    2016-01-01

    Introduction Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Methods and Results Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Conclusions Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP. PMID:27764259

  17. von Willebrand factor fibers promote cancer-associated platelet aggregation in malignant melanoma of mice and humans

    PubMed Central

    Bauer, Alexander T.; Suckau, Jan; Frank, Kathrin; Desch, Anna; Goertz, Lukas; Wagner, Andreas H.; Hecker, Markus; Goerge, Tobias; Umansky, Ludmila; Beckhove, Philipp; Utikal, Jochen; Gorzelanny, Christian; Diaz-Valdes, Nancy; Umansky, Viktor

    2015-01-01

    Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation. PMID:25977583

  18. Red cabbage anthocyanins as inhibitors of lipopolysaccharide-induced oxidative stress in blood platelets.

    PubMed

    Saluk, Joanna; Bijak, Michal; Posmyk, Malgorzata M; Zbikowska, Halina M

    2015-09-01

    LPS is a Gram-negative bacteria endotoxin, which is an important pro-inflammatory agent. Blood platelets take part both in inflammatory processes and in pathogenesis of septic shock following accumulation of LPS. As a platelet agonist LPS causes the intraplatelet overproduction of ROS/RNS that are responsible for adverse modifications in the structure of platelet compounds being associated with a development of platelet-dependent diseases. Existing evidence suggests that anthocyanins (ATH) are able to protect the circulatory system. The antioxidative properties of ATH are believed to be mainly responsible for their positive health effects. The main goal of the present in vitro study was to investigate the potential protective properties of red cabbage ATH against oxidative damage induced by LPS in blood platelets. Exposure of platelets to LPS resulted in carbonyl group increase, 3-nitrotyrosine formation, lipid peroxidation and O2(•-) generation. We have shown that ATH extract effectively decreased oxidative stress induced by LPSs. The in silico analysis demonstrated that both cyanin and LPS were located at the same region of human TLR4-MD-2 complex. Our findings suggest that there could be two-way ATH platelet protection mechanism, by their antioxidant properties and directly by binding with TLRs.

  19. Hypothermia-induced platelet aggregation in human blood in an in vitro model: the dominant role of blood-material interactions.

    PubMed

    Hall, Matthew W; Solen, Kenneth A

    2002-03-05

    Hypothermia-induced platelet aggregation (HIPA) with or without neutrophil involvement may cause neurologic dysfunction during hypothermic surgery. We report the use of a previously developed model to study the contributions of several surfaces, surface area, shear rate, and blood-material exposure time to HIPA. Heparinized (1.5 u/mL) human blood was quenched to 24 degrees C and passed (0.5 mL/min) through a 75-cm long 1/32" ID tubing of polyvinylchloride (PVC), polyethylene (PE), polyurethane (PU), Teflon-FEP, or heparin (Duraflo)-coated PVC. The number of aggregates was measured by a light-scattering method, and the concentration of occlusive aggregates was assessed using constant-pressure filtration (50 mmHg). No differences were seen among PVC, PE, PU, or Teflon-FEP. The heparin-coated PVC tubing produced fewer occlusive aggregates, and heparin leaching from the coating was not the cause of the decrease in occlusive aggregates. Increasing surface area increased the number of aggregates, and increasing shear rates decreased the occlusiveness of those aggregates.

  20. IPA3 non-specifically enhances phosphorylation of several proteins in human platelets.

    PubMed

    Badolia, Rachit; Manne, Bhanu Kanth; Dangelmaier, Carol; Kunapuli, Satya P

    2015-01-01

    PAK (p21-Activation kinase), a serine-threonine protein kinase contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. This autoregulatory domain found within PAK kinase provides a unique target for chemical inhibitors. IPA3, a small molecule allosteric inhibitor of PAK activation, binds covalently to the PAK regulatory domain and prevents binding to its upstream activators. IPA3 has been used in various cells including platelets to evaluate the role of PAK in signaling. In a recent study, PAK functions in platelet aggregation and lamellipodia formation were evaluated using IPA3 as the PAK inhibitor. Herein, we investigated the specificity and selectivity of IPA3 as a PAK inhibitor in the human platelets. Stimulation of platelets pretreated with IPA3 using a PAR-4 or GPV1 agonist resulted in a concentration-dependent inhibition of aggregation, as was suggested by earlier studies. Interestingly, we found that incubation of washed human platelets with IPA3 lead to a non-specific increase in phosphorylation of several proteins in absence of any agonist. However, this phosphorylation is not sufficient for aggregation of platelets by IPA3. In summary, we demonstrate that IPA3 by itself can phosphorylate several proteins in human platelets and thus its use is not an appropriate strategy for investigating PAK function in platelets.

  1. Cholinesterase inhibitors: xanthostigmine derivatives blocking the acetylcholinesterase-induced beta-amyloid aggregation.

    PubMed

    Belluti, Federica; Rampa, Angela; Piazzi, Lorna; Bisi, Alessandra; Gobbi, Silvia; Bartolini, Manuela; Andrisano, Vincenza; Cavalli, Andrea; Recanatini, Maurizio; Valenti, Piero

    2005-06-30

    In continuing research that led us to identify a new class of carbamate derivatives acting as potent (Rampa et al. J. Med. Chem. 1998, 41, 3976) and long-lasting (Rampa et al. J. Med. Chem. 2001, 44, 3810) acetylcholinesterase (AChE) inhibitors, we obtained some analogues able to simultaneously block both the catalytic and the beta-amyloid (Abeta) proaggregatory activities of AChE. The key feature of these derivatives is a 2-arylidenebenzocycloalkanone moiety that provides the ability to bind at the AChE peripheral site responsible for promoting the Abeta aggregation. The new carbamates were tested in vitro for the inhibition of both cholinesterases and also for the ability to prevent the AChE-induced Abeta aggregation. All of the compounds had AChE IC(50) values in the nanomolar range and showed the ability to block the AChE-induced Abeta aggregation, thus supporting the feasibility of this new strategy in the search of compounds for the treatment of Alzheimer's disease.

  2. The influence of prostaglandin G2 on platelet ultrastructure and platelet secretion.

    PubMed Central

    Gerrard, J. M.; Townsend, D.; Stoddard, S.; Witkop, C. J.; White, J. G.

    1977-01-01

    Prostaglandin G2 (PGG2) is a labile endoperoxide produced physiologically following exposure of platelets to aggregating agents. We report here studies using isolated PGG2. This agent stimulates a concentration-dependent internal platelet contraction very similar to that produced by the calcium ionophore A23187. EDTA prevented platelet aggregation but did not prevent PGG2-stimulated internal contraction or secretion. In contrast, prostaglandin E1 and dibutyryl cyclic AMP inhich selectively labilizes platelet granules, was added to platelets together with PGG2 there was a superadditive effect on platelet secretion. Thus, granule labilization induced by PMA is a separable phenomenon and complementary to the effect of PGG2 on contraction. The ultimate degree of secretion is dependent on both processes. Studies using additional inhibitors supported the hypothesis that PGG2 activates platelets (either directly or following conversion to thromboxane A2) by transporting calcium from an intracellular store to the cytoplasmic site of the platelet contractile proteins. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:188341

  3. The effects of aspirin and fish oil consumption on lysophosphatidylcholines and lysophosphatidic acids and their correlates with platelet aggregation in adults with diabetes mellitus

    PubMed Central

    Abdolahi, Amir; Georas, Steve N; Brenna, J. Thomas; Cai, Xueya; Thevenet-Morrison, Kelly; Phipps, Richard P; Lawrence, Peter; Mousa, Shaker A; Block, Robert C

    2014-01-01

    Many diabetics are insensitive to aspirin's platelet anti-aggregation effects. The influence of co-administration of aspirin and fish oil (FO) on plasma lysophospholipids in subjects with diabetes is poorly characterized. Thirty adults with type 2 diabetes mellitus were treated with aspirin (81 mg/day) for seven days, then with FO (4 g/day) for 28 days, then in combination for another seven days. Lysophospholipids and platelet measures were determined after acute (4 hours) and chronic (7 days) ingestion of aspirin, FO, or both in combination. FO ingestion reduced all lysophosphatidic acid (LPA) concentrations, while EPA (20:5n-3) and DHA (22:6n-3) lysophosphatidylcholine (LPC) concentrations significantly increased after FO alone and in combination with aspirin. In vitro arachidonic acid-induced platelet aggregation was most strongly correlated with palmitoleic (16:1) and oleic (18:1) LPA and LPC concentrations at all time points. The ingestion of these agents may reduce cardiovascular disease risk in diabetic adults, with a disrupted lipid milieu, via lysolipid mediated mechanisms. PMID:24373610

  4. Prefoldin Plays a Role as a Clearance Factor in Preventing Proteasome Inhibitor-induced Protein Aggregation*

    PubMed Central

    Abe, Akira; Takahashi-Niki, Kazuko; Takekoshi, Yuka; Shimizu, Takashi; Kitaura, Hirotake; Maita, Hiroshi; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2013-01-01

    Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases. PMID:23946485

  5. 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

  6. Management of pulpal floor perforation and grade II Furcation involvement using mineral trioxide aggregate and platelet rich fibrin: A clinical report.

    PubMed

    Bains, Rhythm; Bains, Vivek K; Loomba, Kapil; Verma, Kavita; Nasir, Afreena

    2012-09-01

    To report the management of an iatrogenic perforation of pulpal floor in the furcation of mandibular first molar, using Mineral Trioxide Aggregate (MTA) and platelet rich fibrin (PRF). Unpredictable endodontic root/pulp chamber floor perforations resulting in unacceptable high rate of clinical failure has now been a lesser threat with the advent of new technologies and biocompatible materials that utilize the applications of basic research along with tissue engineering concept in clinical practice. Present case report illustrates the use of MTA and platelet rich fibrin (PRF) for the repair of the perforation defect and regeneration of the lost periodontium in furcation area. Although, histologic events and reaction of MTA with PRF is not studied so far, however, the autologous and biocompatible nature of the components used for present treatment modalities seems to be beneficial for the long term clinical results obtained in our case.

  7. Management of pulpal floor perforation and grade II Furcation involvement using mineral trioxide aggregate and platelet rich fibrin: A clinical report

    PubMed Central

    Bains, Rhythm; Bains, Vivek K.; Loomba, Kapil; Verma, Kavita; Nasir, Afreena

    2012-01-01

    To report the management of an iatrogenic perforation of pulpal floor in the furcation of mandibular first molar, using Mineral Trioxide Aggregate (MTA) and platelet rich fibrin (PRF). Unpredictable endodontic root/pulp chamber floor perforations resulting in unacceptable high rate of clinical failure has now been a lesser threat with the advent of new technologies and biocompatible materials that utilize the applications of basic research along with tissue engineering concept in clinical practice. Present case report illustrates the use of MTA and platelet rich fibrin (PRF) for the repair of the perforation defect and regeneration of the lost periodontium in furcation area. Although, histologic events and reaction of MTA with PRF is not studied so far, however, the autologous and biocompatible nature of the components used for present treatment modalities seems to be beneficial for the long term clinical results obtained in our case. PMID:23230369

  8. Ramipril and Losartan Exert a Similar Long-Term Effect upon Markers of Heart Failure, Endogenous Fibrinolysis, and Platelet Aggregation in Survivors of ST-Elevation Myocardial Infarction: A Single Centre Randomized Trial

    PubMed Central

    Marinšek, Martin; Sinkovič, Andreja

    2016-01-01

    Introduction. Blocking the renin-angiotensin-aldosterone system in ST-elevation myocardial infarction (STEMI) patients prevents heart failure and recurrent thrombosis. Our aim was to compare the effects of ramipril and losartan upon the markers of heart failure, endogenous fibrinolysis, and platelet aggregation in STEMI patients over the long term. Methods. After primary percutaneous coronary intervention (PPCI), 28 STEMI patients were randomly assigned ramipril and 27 losartan, receiving therapy for six months with dual antiplatelet therapy (DAPT). We measured N-terminal proBNP (NT-proBNP), ejection fraction (EF), plasminogen-activator-inhibitor type 1 (PAI-1), and platelet aggregation by closure times (CT) at the baseline and after six months. Results. Baseline NT-proBNP ≥ 200 pmol/mL was observed in 48.1% of the patients, EF < 55% in 49.1%, and PAI-1 ≥ 3.5 U/mL in 32.7%. Six-month treatment with ramipril or losartan resulted in a similar effect upon PAI-1, NT-proBNP, EF, and CT levels in survivors of STEMI, but in comparison to control group, receiving DAPT alone, ramipril or losartan treatment with DAPT significantly increased mean CT (226.7 ± 80.3 sec versus 158.1 ± 80.3 sec, p < 0.05). Conclusions. Ramipril and losartan exert a similar effect upon markers of heart failure and endogenous fibrinolysis, and, with DAPT, a more efficient antiplatelet effect in long term than DAPT alone. PMID:27064499

  9. Surface modification of CoCr alloy using varying concentrations of phosphoric and phosphonoacetic acids: albumin and fibrinogen adsorption, platelet adhesion, activation, and aggregation studies.

    PubMed

    Thiruppathi, Eagappanath; Larson, Mark K; Mani, Gopinath

    2015-01-01

    CoCr alloy is commonly used in various cardiovascular medical devices for its excellent physical and mechanical properties. However, the formation of blood clots on the alloy surfaces is a serious concern. This research is focused on the surface modification of CoCr alloy using varying concentrations (1, 25, 50, 75, and 100 mM) of phosphoric acid (PA) and phosphonoacetic acid (PAA) to generate various surfaces with different wettability, chemistry, and roughness. Then, the adsorption of blood plasma proteins such as albumin and fibrinogen and the adhesion, activation, and aggregation of platelets with the various surfaces generated were investigated. Contact angle analysis showed PA and PAA coatings on CoCr provided a gradient of hydrophilic surfaces. FTIR showed PA and PAA were covalently bound to CoCr surface and formed different bonding configurations depending on the concentrations of coating solutions used. AFM showed the formation of homogeneous PA and PAA coatings on CoCr. The single and dual protein adsorption studies showed that the amount of albumin and fibrinogen adsorbed on the alloy surfaces strongly depend on the type of PA and PAA coatings prepared by different concentrations of coating solutions. All PA coated CoCr showed reduced platelet adhesion and activation when compared to control CoCr. Also, 75 and 100 mM PA-CoCr showed reduced platelet aggregation. For PAA coated CoCr, no significant difference in platelet adhesion and activation was observed between PAA coated CoCr and control CoCr. Thus, this study demonstrated that CoCr can be surface modified using PA for potentially reducing the formation of blood clots and improving the blood compatibility of the alloy.

  10. Quantitative structure-activity relationship analysis of β-amyloid aggregation inhibitors

    NASA Astrophysics Data System (ADS)

    Stempler, Shiri; Levy-Sakin, Michal; Frydman-Marom, Anat; Amir, Yaniv; Scherzer-Attali, Roni; Buzhansky, Ludmila; Gazit, Ehud; Senderowitz, Hanoch

    2011-02-01

    Inhibiting the aggregation process of the β-amyloid peptide is a promising strategy in treating Alzheimer's disease. In this work, we have collected a dataset of 80 small molecules with known inhibition levels and utilized them to develop two comprehensive quantitative structure-activity relationship models: a Bayesian model and a decision tree model. These models have exhibited high predictive accuracy: 87% of the training and test sets using the Bayesian model and 89 and 93% of the training and test sets, respectively, by the decision tree model. Subsequently these models were used to predict the activities of several new potential β-amyloid aggregation inhibitors and these predictions were indeed validated by in vitro experiments. Key chemical features correlated with the inhibition ability were identified. These include the electro-topological state of carbonyl groups, AlogP and the number of hydrogen bond donor groups. The results demonstrate the feasibility of the developed models as tools for rapid screening, which could help in the design of novel potential drug candidates for Alzheimer's disease.

  11. Spirostanol saponins derivated from the seeds of Trigonella foenum-graecum by β-glucosidase hydrolysis and their inhibitory effects on rat platelet aggregation.

    PubMed

    Pang, Xu; Cong, Yue; Yu, He-Shui; Kang, Li-Ping; Feng, Bing; Han, Bing-Xing; Zhao, Yang; Xiong, Cheng-Qi; Tan, Da-Wei; Song, Wei; Liu, Bin; Cong, Yu-Wen; Ma, Bai-Ping

    2012-02-01

    Nine spirostanol saponins (1-9) and seven mixtures of 25 R and 25 S spirostanol saponin isomers (10-16) were obtained from the seeds of Trigonella foenum-graecum after enzymatic hydrolysis of the furostanol saponin fraction by β-glucosidase. Their structures were determined by NMR and MS spectroscopy. Among them, 1- 4, 6, 8, and 9 were new compounds and five, 11B, 12A, 13B, 14A, and 14B, were new structures observed from seven mixtures. In addition, the inhibitory effects of all saponins on rat platelet aggregation were evaluated.

  12. Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles.

    PubMed Central

    Barry, O. P.; Pratico, D.; Lawson, J. A.; FitzGerald, G. A.

    1997-01-01

    Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells. Activation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction

  13. 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.

  14. Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

    PubMed

    Stokol, Tracy; Serpa, Priscila Beatriz da Silva; Zahid, Muhammad N; Brooks, Marjory B

    2016-01-01

    Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1-0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

  15. Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation

    PubMed Central

    Stokol, Tracy; Serpa, Priscila B. S.; Zahid, Muhammad N.; Brooks, Marjory B.

    2016-01-01

    Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1–0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation

  16. One-step apexification in immature tooth using grey mineral trioxide aggregate as an apical barrier and autologus platelet rich fibrin membrane as an internal matrix

    PubMed Central

    Rudagi, Kavitarani B; Rudagi, BM

    2012-01-01

    Immature teeth with necrotic pulp and periapical lesion are difficult to treat via conventional endodontic therapy. Numerous procedures and materials have been utilized to induce root-end barrier formation. Traditionally, calcium hydroxide has been the material of choice for the apexification of immature permanent teeth; however, Mineral Trioxide Aggregate holds significant promise as an alternative to multiple treatments with calcium hydroxide. One of the technical problems associated with the placement of the restorative materials used as artificial barrier is to prevent overfill and underfill. Using a matrix avoids the extrusion of the material into the periodontal tissues. This case report presents the successful healing and apexification with combined use of Mineral Trioxide Aggregate as an apical barrier, and autologus platelet rich fibrin membrane as an internal matrix. PMID:22557824

  17. Complete Phenotypic Recovery of an Alzheimer's Disease Model by a Quinone-Tryptophan Hybrid Aggregation Inhibitor

    PubMed Central

    Scherzer-Attali, Roni; Pellarin, Riccardo; Convertino, Marino; Frydman-Marom, Anat; Egoz-Matia, Nirit; Peled, Sivan; Levy-Sakin, Michal; Shalev, Deborah E.; Caflisch, Amedeo; Gazit, Ehud; Segal, Daniel

    2010-01-01

    The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease. PMID:20559435

  18. A novel aggregation-induced emission based fluorescent probe for an angiotensin converting enzyme (ACE) assay and inhibitor screening.

    PubMed

    Wang, Haibo; Huang, Yi; Zhao, Xiaoping; Gong, Wan; Wang, Yi; Cheng, Yiyu

    2014-12-11

    A 'turn-on' fluorescent probe based on aggregation-induced emission (AIE) has been developed. It exhibits excellent selectivity and sensitivity for monitoring angiotensin converting enzyme (ACE) activity both in solutions and in living cells as well as for screening ACE inhibitors in vitro.

  19. 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.

  20. 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.

  1. Membranoproliferative glomerulonephritis. A prospective clinical trial of platelet-inhibitor therapy

    SciTech Connect

    Donadio, J.V. Jr.; Anderson, C.F.; Mitchell, J.C.; Holley, K.E.; Ilstrup, D.M.; Fuster, V.; Chesebro, J.H.

    1984-05-31

    Forty patients with Type I membranoproliferative glomerulonephritis were treated for one year with dipyridamole, 225 mg per day, and aspirin, 975 mg per day, in a prospective, randomized, double-blind, placebo-controlled study. At the base line, the half-life of /sup 51/Cr-labeled platelets was reduced in 12 of 17 patients. The platelet half-life became longer and renal function stabilized in the treated group, as compared with the placebo group, suggesting a relation between platelet consumption and the glomerulopathy. The glomerular filtration rate, determined by iothalamate clearance, was better maintained in the treated group (average decrease, 1.3 ml per minute per 1.73 m/sup 2/ of body-surface area per 12 months) than in the placebo group (average decrease, 19.6). Fewer patients in the treated group than in the placebo group had progression to end-stage renal disease (3 of 21 after 62 months as compared with 9 of 19 after 33 months). The data suggest that dipyridamole and aspirin slowed the deterioration of renal function and the development of end-stage renal disease.

  2. Isolation of intact megakaryocytes from guinea pig femoral marrow. Successful harvest made possible with inhibitions of platelet aggregation; enrichment achieved with a two-step separation technique

    PubMed Central

    1976-01-01

    Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro. PMID:3509

  3. 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.

  4. Selective Inhibition of ADAM17 Efficiently Mediates Glycoprotein Ibα Retention During Ex Vivo Generation of Human Induced Pluripotent Stem Cell-Derived Platelets.

    PubMed

    Hirata, Shinji; Murata, Takahiko; Suzuki, Daisuke; Nakamura, Sou; Jono-Ohnishi, Ryoko; Hirose, Hidenori; Sawaguchi, Akira; Nishimura, Satoshi; Sugimoto, Naoshi; Eto, Koji

    2017-03-01

    Donor-independent platelet concentrates for transfusion can be produced in vitro from induced pluripotent stem cells (iPSCs). However, culture at 37°C induces ectodomain shedding on platelets of glycoprotein Ibα (GPIbα), the von Willebrand factor receptor critical for adhesive function and platelet lifetime in vivo, through temperature-dependent activation of a disintegrin and metalloproteinase 17 (ADAM17). The shedding can be suppressed by using inhibitors of panmetalloproteinases and possibly of the upstream regulator p38 mitogen-activated protein kinase (p38 MAPK), but residues of these inhibitors in the final platelet products may be accompanied by harmful risks that prevent clinical application. Here, we optimized the culture conditions for generating human iPSC-derived GPIbα(+) platelets, focusing on culture temperature and additives, by comparing a new and safe selective ADAM17 inhibitor, KP-457, with previous inhibitors. Because cultivation at 24°C (at which conventional platelet concentrates are stored) markedly diminished the yield of platelets with high expression of platelet receptors, 37°C was requisite for normal platelet production from iPSCs. KP-457 blocked GPIbα shedding from iPSC platelets at a lower half-maximal inhibitory concentration than panmetalloproteinase inhibitor GM-6001, whereas p38 MAPK inhibitors did not. iPSC platelets generated in the presence of KP-457 exhibited improved GPIbα-dependent aggregation not inferior to human fresh platelets. A thrombus formation model using immunodeficient mice after platelet transfusion revealed that iPSC platelets generated with KP-457 exerted better hemostatic function in vivo. Our findings suggest that KP-457, unlike GM-6001 or p38 MAPK inhibitors, effectively enhances the production of functional human iPSC-derived platelets at 37°C, which is an important step toward their clinical application. Stem Cells Translational Medicine 2017;6:720-730.

  5. Succinate reverses in-vitro platelet inhibition by acetylsalicylic acid and P2Y receptor antagonists.

    PubMed

    Spath, Brigitte; Hansen, Arne; Bokemeyer, Carsten; Langer, Florian

    2012-01-01

    High on-treatment platelet reactivity has been associated with adverse cardiovascular events in patients receiving anti-platelet agents, but the molecular mechanisms underlying this phenomenon remain incompletely understood. Succinate, a citric acid cycle intermediate, is released into the circulation under conditions of mitochondrial dysfunction due to hypoxic organ damage, including sepsis, stroke, and myocardial infarction. Because the G protein-coupled receptor (GPCR) for succinate, SUCNR1 (GPR91), is present on human platelets, we hypothesized that succinate-mediated platelet stimulation may counteract the pharmacological effects of cyclooxygenase-1 and ADP receptor antagonists. To test this hypothesis in a controlled in-vitro study, washed platelets from healthy donors were treated with acetylsalicylic acid (ASA) or small-molecule P2Y(1) or P2Y(12) inhibitors and subsequently analyzed by light transmittance aggregometry using arachidonic acid (AA), ADP and succinate as platelet agonists. Aggregation in response to succinate alone was highly variable with only 29% of donors showing a (mostly delayed) platelet response. In contrast, succinate reproducibly and concentration-dependently (10-1000 µM) enhanced platelet aggregation in response to low concentrations of exogenous ADP. Furthermore, while succinate alone had no effect in the presence of platelet inhibitors, responsiveness of platelets to ADP after pretreatment with P2Y(1) or P2Y(12) antagonists was fully restored, when platelets were co-stimulated with 100 µM succinate. Similarly, succinate completely (at 1000 µM) or partially (at 100 µM) reversed the inhibitory effect of ASA on AA-induced platelet aggregation. In contrast, succinate failed to restore platelet responsiveness in the presence of both ASA and the P2Y(12) antagonist, suggesting that concomitant signaling via different GPCRs was required. Essentially identical results were obtained, when flow cytometric analysis of surface CD62P

  6. Acetylsalicylic Acid Daily vs Acetylsalicylic Acid Every 3 Days in Healthy Volunteers: Effect on Platelet Aggregation, Gastric Mucosa, and Prostaglandin E2 Synthesis.

    PubMed

    Ferreira, Plinio Minghin Freitas; Gagliano-Jucá, Thiago; Zaminelli, Tiago; Sampaio, Marinalva Ferreira; Blackler, Rory Willian; Trevisan, Miriam da Silva; Novaes Magalhães, Antônio Frederico; De Nucci, Gilberto

    2016-07-01

    Substantial platelet inhibition was observed 3 days after a single administration of acetylsalicylic acid 81 mg to healthy volunteers. Here we investigate prostaglandin E2 (PGE2 ) antrum concentrations and gastrointestinal symptoms in two treatment groups: one receiving losartan and acetylsalicylic acid every day and the other receiving losartan every day and acetylsalicylic acid every 3 days. Twenty-eight healthy volunteers from both sexes received either 50 mg losartan and acetylsalicylic acid 81 mg daily or 50 mg losartan and acetylsalicylic acid 81 every 3 days with placebo on the other days. Therapy was delivered for 30 days for both groups. Gastric endoscopy was performed before and after treatment period. Biopsies were collected for PGE2 quantification. Platelet function tests were carried out before and during treatment and TXB2 release on platelet rich plasma was measured. The every 3 day low-dose acetylsalicylic acid regimen produced complete inhibition of platelet aggregation compared to the daily treatment. Thromboxane B2 release was substantially abolished for both groups during treatment. There was no significant difference on the endoscopic score of both treatment groups after the 30-day treatment (P = .215). There was over 50% suppression of antrum PGE2 content on volunteers receiving acetylsalicylic acid daily (P = .0016), while for the every 3 day dose regimen there was no significant difference between pre and post-treatment antrum PGE2 dosages (P = .4193). Since PGE2 is involved in gastric healing, we understand that this new approach could be safer and as efficient as the standard daily therapy on a long-term basis.

  7. Dual inhibitors of β-amyloid aggregation and acetylcholinesterase as multi-target anti-Alzheimer drug candidates.

    PubMed

    Viayna, Elisabet; Sabate, Raimon; Muñoz-Torrero, Diego

    2013-01-01

    Notwithstanding the functional role that the aggregates of some amyloidogenic proteins can play in different organisms, protein aggregation plays a pivotal role in the pathogenesis of a large number of human diseases. One of such diseases is Alzheimer's disease (AD), where the overproduction and aggregation of the β-amyloid peptide (Aβ) are regarded as early critical factors. Another protein that seems to occupy a prominent position within the complex pathological network of AD is the enzyme acetylcholinesterase (AChE), with classical and non-classical activities involved at the late (cholinergic deficit) and early (Aβ aggregation) phases of the disease. Dual inhibitors of Aβ aggregation and AChE are thus emerging as promising multi-target agents with potential to efficiently modify the natural course of AD. In the initial phases of the drug discovery process of such compounds, in vitro evaluation of the inhibition of Aβ aggregation is rather troublesome, as it is very sensitive to experimental assay conditions, and requires expensive synthetic Aβ peptides, which makes cost-prohibitive the screening of large compound libraries. Herein, we review recently developed multitarget anti-Alzheimer compounds that exhibit both Aβ aggregation and AChE inhibitory activities, and, in some cases also additional valuable activities such as BACE-1 inhibition or antioxidant properties. We also discuss the development of simplified in vivo methods for the rapid, simple, reliable, unexpensive, and high-throughput amenable screening of Aβ aggregation inhibitors that rely on the overexpression of Aβ42 alone or fused with reporter proteins in Escherichia coli.

  8. Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

    PubMed

    Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2015-06-01

    The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis.

  9. Coagulation factor XI promotes distal platelet activation and single platelet consumption in the bloodstream under shear flow

    PubMed Central

    Zilberman-Rudenko, Jevgenia; Itakura, Asako; Wiesenekker, Chantal P.; Vetter, Ralf; Maas, Coen; Gailani, David; Tucker, Erik I.; Gruber, András; Gerdes, Christoph; McCarty, Owen J. T.

    2016-01-01

    Objective Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal thrombus formation without affecting proximal platelet adhesion. PMID:26769048

  10. Identification of selective covalent inhibitors of platelet activating factor acetylhydrolase 1B2 from the screening of an oxadiazolone-capped peptoid-azapeptoid hybrid library.

    PubMed

    Sarma, Bani Kanta; Liu, Xiaodan; Kodadek, Thomas

    2016-09-01

    A potent and selective inhibitor of platelet-activating factor acetylhydrolase 1B2 (PAFAH1B2) is described. The compound was derived by improvement of a modest affinity primary hit isolated from the screening of a bead-displayed peptoid-azapeptoid hybrid library tethered to an oxadiazolone 'warhead'. The oxadiazolone moiety of the inhibitors was found to react covalently with the active site serine residue of PAFAH1B2. This screening strategy may be useful for the identification of many selective, covalent inhibitors of serine hydrolases.

  11. Lower-Extremity Functional Electrical Stimulation Decreases Platelet Aggregation and Blood Coagulation in Persons With Chronic Spinal Cord Injury: A Pilot Study

    PubMed Central

    Kahn, Nighat N; Feldman, Susan P; Bauman, William A

    2010-01-01

    Background: Individuals with spinal cord injury (SCI) develop premature cardiovascular disease. Regular exercise reduces the incidence and symptoms of cardiovascular disease in able-bodied individuals; these salutary effects of exercise have not been documented in persons with SCI. Objective: To evaluate the effects of functional electrical stimulation leg cycle ergometry (FES-LCE) exercise training on platelet aggregation and blood coagulation in persons with SCI. Participants: Subjects (n  =  14) with stable chronic (>1 year) paraplegia (T1–T10) or tetraplegia (C4–C8). Methods: Blood samples were collected before and after the first and eighth sessions (2 sessions per week for 4 weeks) of FES exercise. Results: Platelet aggregation was inhibited by 20% after the first session and by 40% (P < 0.001) after the eighth session. Thrombin activity was unchanged after the first session (10.7 ± 0.85 s to 10.43 ± 0.56 s) and decreased after the eighth session (12.5 ± 1.98 s to 11.1 ± 1.7 s; P < 0.0003). Antithrombin III activity increased after the first (103.8% ± 8.9% to 110% ± 6.9%; P < 0.0008) and eighth sessions (107.8% ± 12.1% to 120.4% ± 13.1%; P < 0.0001). Cyclic adenosine monophosphate increased after the first (9.9% ± 2.5% to 15.8% ± 3%; P < 0.001) and eighth sessions (17.8% ± 4.2% to 36.5% ± 7.6%; P < 0.0001). After the eighth session, factors V and X increased significantly (88% ± 27% to 103% ± 23%, P < 0.0001; 100% ± 40% to 105% ± 7%, P < 0.01, respectively); factors VII and VIII and fibrinogen did not change significantly. A significant reduction in platelet activation/aggregation was demonstrated in response to FES-LCE. The decrease in thrombin level was caused by the simultaneous increase in antithrombin activity. Conclusion: These findings provide new insight into the potential protective effects of FES-LCE against the risk of cardiovascular disease. PMID:20486534

  12. Piperlongumine Blocks JAK2-STAT3 to Inhibit Collagen-Induced Platelet Reactivity Independent of Reactive Oxygen Species†

    PubMed Central

    Yuan, Hengjie; Houck, Katie L.; Tian, Ye; Bharadwaj, Uddalak; Hull, Ken; Zhou, Zhou; Zhou, Mingzhao; Wu, Xiaoping; Tweardy, David J.; Romo, Daniel; Fu, Xiaoyun; Zhang, Yanjun; Zhang, Jianning; Dong, Jing-fei

    2015-01-01

    Background Piperlongumine (PL) is a compound isolated from the piper longum plant. It possesses anti-cancer activities through blocking the transcription factor STAT3 and by inducing reactive oxygen species (ROS) in cancer, but not normal cells. It also inhibits platelet aggregation induced by collagen, but the underlying mechanism is not known. Objective We conducted in vitro experiments to test the hypothesis that PL regulates a non-transcriptional activity of STAT3 to specifically reduce the reactivity of human platelets to collagen. Results PL dose-dependently blocked collagen-induced platelet aggregation, calcium influx, CD62p expression and thrombus formation on collagen with a maximal inhibition at 100 μM. It reduced platelet microvesiculation induced by collagen. PL blocked the activation of JAK2 and STAT3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets pretreated with a STAT3 inhibitor. Although PL induced ROS production in platelets; quenching ROS using excessive reducing agents: 20 μM GSH and 0.5 mM L-Cysteine, did not block the inhibitory effects. The NADPH oxidase inhibitor Apocynin also had no effect. Conclusions PL inhibited collagen-induced platelet reactivity by targeting the JAK2-STAT3 pathway. We also provide experimental evidence that PL and collagen induce different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants in redox signals. PMID:26645674

  13. Interaction of blood platelets with a microfibrillar extract from adult bovine aorta: requirement for von Willebrand factor.

    PubMed Central

    Fauvel, F; Grant, M E; Legrand, Y J; Souchon, H; Tobelem, G; Jackson, D S; Caen, J P

    1983-01-01

    Adult bovine aortic tissue was treated with 6 M guanidinium chloride in the presence of proteinase inhibitors to obtain an extract that was essentially devoid of collagenous components and appeared homogeneous by electron microscopy. When this extract was dispersed by sonication it was found to be a very potent inducer of human platelet aggregation. This interaction required the presence of von Willebrand factor and of its receptor (glycoprotein Ib) on platelet membrane. This was demonstrated by the fact that the aggregation of normal blood platelets resuspended in plasmas deficient in von Willebrand factor was significantly diminished as compared to aggregation in control plasma. Moreover, this aggregation was inhibited by a monoclonal antibody, IgG AN51, to platelet glycoprotein Ib. These studies provide direct biochemical evidence for the existence of a thrombogenic constituent of the vessel wall that is noncollagenous and von Willebrand factor-dependent. Images PMID:6601274

  14. Platelets protect lung from injury induced by systemic inflammatory response

    PubMed Central

    Luo, Shuhua; Wang, Yabo; An, Qi; Chen, Hao; Zhao, Junfei; Zhang, Jie; Meng, Wentong; Du, Lei

    2017-01-01

    Systemic inflammatory responses can severely injure lungs, prompting efforts to explore how to attenuate such injury. Here we explored whether platelets can help attenuate lung injury in mice resulting from extracorporeal circulation (ECC)-induced systemic inflammatory responses. Mice were subjected to ECC for 30 min, then treated with phosphate-buffered saline, platelets, the GPIIb/IIIa inhibitor Tirofiban, or the combination of platelets and Tirofiban. Blood and lung tissues were harvested 60 min later, and lung injury and inflammatory status were assessed. As expected, ECC caused systemic inflammation and pulmonary dysfunction, and platelet transfusion resulted in significantly milder lung injury and higher lung function. It also led to greater numbers of circulating platelet-leukocyte aggregates and greater platelet accumulation in the lung. Platelet transfusion was associated with higher production of transforming growth factor-β and as well as lower levels of tumour necrosis factor-α and neutrophil elastase in plasma and lung. None of these platelet effects was observed in the presence of Tirofiban. Our results suggest that, at least under certain conditions, platelets can protect lung from injury induced by systemic inflammatory responses. PMID:28155889

  15. Thioflavin-S staining of bacterial inclusion bodies for the fast, simple, and inexpensive screening of amyloid aggregation inhibitors.

    PubMed

    Pouplana, S; Espargaro, A; Galdeano, C; Viayna, E; Sola, I; Ventura, S; Muñoz-Torrero, D; Sabate, R

    2014-01-01

    Amyloid aggregation is linked to a large number of human disorders, from neurodegenerative diseases as Alzheimer's disease (AD) or spongiform encephalopathies to non-neuropathic localized diseases as type II diabetes and cataracts. Because the formation of insoluble inclusion bodies (IBs) during recombinant protein production in bacteria has been recently shown to share mechanistic features with amyloid self-assembly, bacteria have emerged as a tool to study amyloid aggregation. Herein we present a fast, simple, inexpensive and quantitative method for the screening of potential anti-aggregating drugs. This method is based on monitoring the changes in the binding of thioflavin-S to intracellular IBs in intact Eschericchia coli cells in the presence of small chemical compounds. This in vivo technique fairly recapitulates previous in vitro data. Here we mainly use the Alzheimer's related β-amyloid peptide as a model system, but the technique can be easily implemented for screening inhibitors relevant for other conformational diseases simply by changing the recombinant amyloid protein target. Indeed, we show that this methodology can be also applied to the evaluation of inhibitors of the aggregation of tau protein, another amyloidogenic protein with a key role in AD.

  16. Inhibitory effects of Tabebuia impetiginosa inner bark extract on platelet aggregation and vascular smooth muscle cell proliferation through suppressions of arachidonic acid liberation and ERK1/2 MAPK activation.

    PubMed

    Son, Dong-Ju; Lim, Yong; Park, Young-Hyun; Chang, Sung-Keun; Yun, Yeo-Pyo; Hong, Jin-Tae; Takeoka, Gary R; Lee, Kwang-Geun; Lee, Sung-Eun; Kim, Mi-Ran; Kim, Jeong-Han; Park, Byeoung-Soo

    2006-11-03

    The antiplatelet and antiproliferative activities of extract of Tabebuia impetiginosa inner bark (taheebo) were investigated using washed rabbit platelets and cultured rat aortic vascular smooth muscle cells (VSMCs) in vitro. n-Hexane, chloroform and ethyl acetate fractions showed marked and selective inhibition of platelet aggregation induced by collagen and arachidonic acid (AA) in a dose-dependent manner. These fractions, especially the chloroform fraction, also significantly suppressed AA liberation induced by collagen in [(3)H]AA-labeled rabbit platelets. The fractions, especially the chloroform fraction, potently inhibited cell proliferation and DNA synthesis induced by platelet derived growth factor (PDGF)-BB, and inhibited the levels of phosphorylated extracellular signal regulated kinase (ERK1/2) mitogen activated protein kinase (MAPK) stimulated by PDGF-BB, in the same concentration range that inhibits VSMC proliferation and DNA synthesis.

  17. The P2Y12 antagonists, 2-methylthioadenosine 5'-monophosphate triethylammonium salt and cangrelor (ARC69931MX), can inhibit human platelet aggregation through a Gi-independent increase in cAMP levels.

    PubMed

    Srinivasan, Subhashini; Mir, Fozia; Huang, Jin-Sheng; Khasawneh, Fadi T; Lam, Stephen C-T; Le Breton, Guy C

    2009-06-12

    ADP plays an integral role in the process of hemostasis by signaling through two platelet G-protein-coupled receptors, P2Y1 and P2Y12. The recent use of antagonists against these two receptors has contributed a substantial body of data characterizing the ADP signaling pathways in human platelets. Specifically, the results have indicated that although P2Y1 receptors are involved in the initiation of platelet aggregation, P2Y12 receptor activation appears to account for the bulk of the ADP-mediated effects. Based on this consideration, emphasis has been placed on the development of a new class of P2Y12 antagonists (separate from clopidogrel and ticlopidine) as an approach to the treatment of thromboembolic disorders. The present work examined the molecular mechanisms by which two of these widely used adenosine-based P2Y12 antagonists (2-methylthioadenosine 5'-monophosphate triethylammonium salt (2MeSAMP) and ARC69931MX), inhibit human platelet activation. It was found that both of these compounds raise platelet cAMP to levels that substantially inhibit platelet aggregation. Furthermore, the results demonstrated that this elevation of cAMP did not require Gi signaling or functional P2Y12 receptors but was mediated through activation of a separate G protein-coupled pathway, presumably involving Gs. However, additional experiments revealed that neither 2MeSAMP nor ARC69931MX (cangrelor) increased cAMP through activation of A2a, IP, DP, or EP2 receptors, which are known to couple to Gs. Collectively, these findings indicate that 2MeSAMP and ARC69931MX interact with an unidentified platelet G protein-coupled receptor that stimulates cAMP-mediated inhibition of platelet function. This inhibition is in addition to that derived from antagonism of P2Y12 receptors.

  18. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    PubMed Central

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  19. A Pilot Study on the Effect of Angiotensin Receptor Blockers on Platelet Aggregation in Hypertensive Patients- A Prospective Observational Study

    PubMed Central

    Sanji, Narendranath; Kamath, Pallavi Mahadeva; Devendrappa, Srinivas Lokikere; Hanumanthareddy, Shashikala Gowdara; Maniyar, Imran; Rudrappa, Suresh Surappla

    2016-01-01

    Introduction Thrombosis is an invariable component contributing to cardiovascular events in patients with hypertension. One of the risk factors of cardiovascular disease is increased platelet activity. One among the widely used antihypertensive agents are Angiotensin II type 1 Receptor Blockers (ARBs). Even though there are many studies involving antihypertensive agents, their antithrombotic properties remain elusive and not fully characterized. Aim To evaluate the anti-aggregatory effect of ARBs on platelets in-vivo. Materials and Methods A total of 60 subjects were included in this observational pilot study conducted in the medicine out patient department of JJM Hospital, Davanagere, Karnataka, India. Among them, 30 patients with essential hypertension attending Medicine OPD of a tertiary care hospital, who were on ARB for at least one month, were enrolled into study group. The control group consisted of 30 normotensive subjects who were not on any drug affecting platelet function. The Bleeding Time (BT) was evaluated for both the groups using Duke method of BT estimation. Data was analysed using SPSS software version 20. The test group was compared with control group using student’s unpaired t-test. Results The mean BT of study group was 2.488 minutes ± 0.0361 Standard Error of Mean (SEM) and that of control group was 1.998 minutes ± 0.0362 SEM. The result was statistically significant (p<0.001). The average duration of treatment was 2.933 years. Conclusion ARB have antiplatelet activity. Increase in BT in ARB group when compared with that of control group is a reflection of antiplatelet activity. PMID:28050394

  20. The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine.

    PubMed

    Nylander, M; Lindahl, T L; Bengtsson, T; Grenegård, M

    2008-08-01

    Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct

  1. 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.

  2. 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.

  3. Study of a Bifunctional Aβ Aggregation Inhibitor with the Abilities of Antiamyloid-β and Copper Chelation.

    PubMed

    Zhang, Qian; Hu, Xiaoyu; Wang, Wei; Yuan, Zhi

    2016-02-08

    In this study, a bifunctional Aβ aggregation inhibitor peptide, GGHRYYAAFFARR (GR), with the abilities to bind copper and antiamyloid was designed to inhibit the neurotoxicity of the Aβ-Cu(II) complex. The thioflavin T (ThT) assay, turbidimetric analysis, transmission electron microscopy (TEM), and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay were used to study its potential inhibitory effect on Aβ aggregation. Our findings indicate that GGH was the specific chelating sequence and that the RYYAAFFARR (RR) component acted as an aggregation inhibitor. More importantly, GR significantly decreased the cytotoxicity of the Aβ-Cu(II) complex. The cell viability improved to 88%, which was higher than with the single functional peptide GGH and RR by 39% and 20%, respectively. Moreover, the qualitative effect of Cu(II) on the Aβ-Cu(II) complex was also studied. Our results indicate that Cu(II) induces the formation of the β-sheet structure with a subequimolar Cu(II):Aβ molar ratio (0.25:1) but led to increased ROS production at a supra-equimolar ratio.

  4. Effect of some cyclooxygenase inhibitors on the increase in guanosine 3′:5′-cyclic monophosphate induced by NO-donors in human whole platelets

    PubMed Central

    Failli, Paola; Cecchi, Enrica; Tosti-Guerra, Cristina; Mugelli, Alessandro; Laffi, Giacomo; Zilletti, Lucilla; Giotti, Alberto

    1998-01-01

    The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3′:5′-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate.In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC50 being 13.7 μM and 15.8 μM, respectively.Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC50=32.7 μM, NaNP and 25.0 μM, SNAP), while acetylsalicylic acid (up to 1000 μM) and diclofenac (up to 100 μM) were ineffective.However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production.Indomethacin (10 μM), indoprofen (30 μM), diclofenac (100 μM) and acetylsalicylic acid (1000 μM) showed a comparable efficacy in inhibiting platelet thromboxane B2 (TXB2) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase.In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP.The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen. PMID:9579743

  5. Ruthenium(II) polypyridyl complex as inhibitor of acetylcholinesterase and Aβ aggregation.

    PubMed

    Vyas, Nilima A; Bhat, Satish S; Kumbhar, Avinash S; Sonawane, Uddhavesh B; Jani, Vinod; Joshi, Rajendra R; Ramteke, Shefali N; Kulkarni, Prasad P; Joshi, Bimba

    2014-03-21

    Two ruthenium(II) polypyridyl complexes [Ru(phen)3](2+) (1) and [Ru(phen)2(bxbg)](2+) (2) (where phen = 1,10 phenanthroline, bxbg = bis(o-xylene)bipyridine glycoluril) have been evaluated for acetylcholinesterase (AChE) and Amyloid-β peptide (Aβ) aggregation inhibition. Complex 2 exhibits higher potency of AChE inhibition and kinetics and molecular modeling studies indicate that ancillary ligand plays significant role in inhibitory potency exhibited by complex 2. The inhibitory effect of these complexes on Aβ (1-40) aggregation is investigated using Thioflavin T fluorescence and Transmission Electron Microscopy. Both complexes efficiently inhibit Aβ (1-40) aggregation and are negligibly toxic to human neuroblastoma cells. This is the first demonstration that ruthenium(II) polypyridyl complexes simultaneously inhibit AChE and Aβ aggregation.

  6. 21 CFR 864.6675 - Platelet aggregometer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675...

  7. 21 CFR 864.6675 - Platelet aggregometer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... shape and platelet aggregation following the addition of an aggregating reagent to a platelet rich... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Platelet aggregometer. 864.6675 Section 864.6675...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6675...

  8. Human Platelets Recognize a Novel Surface Protein, PadA, on Streptococcus gordonii through a Unique Interaction Involving Fibrinogen Receptor GPIIbIIIa▿ †

    PubMed Central

    Petersen, Helen J.; Keane, Ciara; Jenkinson, Howard F.; Vickerman, M. Margaret; Jesionowski, Amy; Waterhouse, Janet C.; Cox, Dermot; Kerrigan, Steven W.

    2010-01-01

    The concept of an infectious agent playing a role in cardiovascular disease is slowly gaining attention. Among several pathogens identified, the oral bacterium Streptococcus gordonii has been implicated as a plausible agent. Platelet adhesion and subsequent aggregation are critical events in the pathogenesis and dissemination of the infective process. Here we describe the identification and characterization of a novel cell wall-anchored surface protein, PadA (397 kDa), of S. gordonii DL1 that binds to the platelet fibrinogen receptor GPIIbIIIa. Wild-type S. gordonii cells induced platelet aggregation and supported platelet adhesion in a GPIIbIIIa-dependent manner. Deletion of the padA gene had no effect on platelet aggregation by S. gordonii but significantly reduced (>75%) platelet adhesion to S. gordonii. Purified N-terminal PadA recombinant polypeptide adhered to platelets. The padA mutant was unaffected in production of other platelet-interactive surface proteins (Hsa, SspA, and SspB), and levels of adherence of the mutant to fetuin or platelet receptor GPIb were unaffected. Wild-type S. gordonii, but not the padA mutant, bound to Chinese hamster ovary cells stably transfected with GPIIbIIIa, and this interaction was ablated by addition of GPIIbIIIa inhibitor Abciximab. These results highlight the growing complexity of interactions between S. gordonii and platelets and demonstrate a new mechanism by which the bacterium could contribute to unwanted thrombosis. PMID:19884334

  9. 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

  10. An effect of glycoprotein IIb/IIIa inhibitors on the kinetics of red blood cells aggregation.

    PubMed

    Sokolova, Irina A; Muravyov, Alexei V; Khokhlova, Maria D; Rikova, Sofya Yu; Lyubin, Evgeny V; Gafarova, Marina A; Skryabina, Maria N; Fedyanin, Angrey A; Kryukova, Darya V; Shahnazarov, Alexander A

    2014-01-01

    The reversible aggregation of red blood cells (RBCs) continues to be of the basic science and clinical interest. Recently it has been reported about a specific binding between fibrinogen and unknown erythrocyte glycoprotein receptors. The aim of this study was to investigate whether the red blood cell aggregation (RBCA) include the cell-cell interaction using the membrane receptors that bind such ligands as fibrinogen or fibronectin. To test this hypothesis the RBCs were incubated with monafram - the drug of the monoclonal antibodies against glycoprotein (GP) IIb/IIIa, with the GPIIb-IIIa receptor antagonist tirofiban, epifibatide and with the fibrinogen inhibiting peptide. It has been found that the RBC incubation with monafram resulted in a marked RBCA decrease mainly in persons with high level of aggregation. Another research session has shown that RBC incubation with fibronectin was accompanied by a significant RBCA rise. The monafram addition to red cell incubation medium resulted in a significant RBCA lowering. The cell incubation with tirofiban and epifibatide issued in RBCA decrease. The similar results were obtained when RBCs were incubated with the fibrinogen inhibiting peptide. Although monafram, tirofiban, eptifibatide and the fibrinogen inhibiting peptide were related to fibrinogen function they didn't inhibit RBCA completely. Therefore, under moderate and low red blood cell aggregation the cell binding is probably related to nonspecific mode. It seems evident that the specific and nonspecific modes of red blood cell aggregate formation could co-exist. Additional theoretical and experimental investigations in this area are needed.

  11. PK10453, a nonselective platelet-derived growth factor receptor inhibitor, prevents the progression of pulmonary arterial hypertension

    PubMed Central

    2014-01-01

    Abstract The platelet-derived growth factor (PDGF) signaling pathway has been found to be activated in human pulmonary arterial hypertension (PAH) and in animal models of the disease. Our study tested the hypothesis that a novel, nonselective inhaled PDGF receptor inhibitor, PK10453, would decrease pulmonary hypertension both in the rat monocrotaline (MCT) model and the rat MCT plus pneumonectomy (MCT+PN) model of PAH. PK10453, delivered by inhalation for 4 (D4)- and 8 (D8)-minute exposures 3 times a day for 2 weeks, decreased right ventricular systolic pressure (RVSP) in both the rat MCT and rat MCT+PN models: RVSP was 80.4 ± 2.6 mmHg in the vehicle MCT group (n = 6), 44.4 ± 5.8 mmHg in the D4 MCT group (n = 6), and 37.1 ± 4.5 mmHg in the D8 MCT group (n = 5; P < 0.001 vs. vehicle); RVSP was 75.7 ± 7.1 mmHg in the vehicle MCT+PN group (n = 9), 40.4 ± 2.7 mmHg in the D4 MCT+PN group (n = 10), and 43.0 ± 3.0 mmHg in the D8 MCT+PN group (n = 8; P < 0.001). In the rat MCT+PN model, continuous telemetry monitoring of pulmonary artery pressures also demonstrated that PK10453 prevented the progression of PAH. Imatinib given by inhalation was equally effective in the MCT model but was not effective in the MCT+PN model. Immunohistochemistry demonstrated increased activation of the PDGFβ receptor compared to the PDGFα receptor in neointimal and perivascular lesions found in the MCT+PN model. We show that imatinib is selective for the PDGFα receptor, whereas PK10453 has a lower half-maximal inhibitor concentration (IC50) for inhibition of kinase activity of both the PDGFα and PDGFβ receptors compared to imatinib. In conclusion, PK10453, when delivered by inhalation, significantly decreased the progression of PAH in the rat MCT and MCT+PN models. Nonselective inhibition of both the PDGFα and PDGFβ receptors may have a therapeutic advantage over selective PDGFα receptor inhibition in PAH. PMID:25006424

  12. Cisplatin triggers platelet activation.

    PubMed

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

    2000-09-01

    Clinical observations suggest that anticancer drugs could contribute to the thrombotic complications of malignancy in treated patients. Thrombotic microangiopathy, myocardial infarction, and cerebrovascular thrombotic events have been reported for cisplatin, a drug widely used in the treatment of many solid tumours. The aim of this study is to explore in vitro cisplatin effect on human platelet reactivity in order to define the potentially active role of platelets in the pathogenesis of cisplatin-induced thrombotic complications. Our results demonstrate that cisplatin increases human platelet reactivity (onset of platelet aggregation wave and thromboxane production) to non-aggregating concentrations of the agonists involving arachidonic acid metabolism. Direct or indirect activation of platelet phospholipase A(2) appears to be implicated. This finding contributes to a better understanding of the pathogenesis of thrombotic complications occurring during cisplatin-based chemotherapy.

  13. Local shear conditions and platelet aggregates regulate the incorporation and activity of circulating tissue factor in ex-vivo thrombi.

    PubMed

    Balasubramanian, Viji; Vele, Oana; Nemerson, Yale

    2002-11-01

    The presence of thrombogenic blood-borne or circulating tissue factor (cTF) has recently been demonstrated. These observations have implicated cTF to be a key determinant of thrombus propagation by depositing on platelets in nascent thrombi. Previously, we detected cTF by detergent solubilization and addition of phospholipids. We now report the direct demonstration of TF activity in ex-vivo thrombi. Collagen-coated substrates were exposed to native blood at shear rates of 0, 650, and 2,000 s(-1) for 10 min in a modified rotating Teflon cone and plate viscometer. Substrates were then gently rinsed to remove 'loose' (unadherent) components of blood. cTF activity was measured by adding a solution containing 10 nM FVIIa, 100 nM FX, and 5 mM CaCl(2) to the substrates exposed to blood. Samples of this mixture were obtained at intervals for 30 min and the amount of Xa generated was quantified by adding a chromogenic substrate, Spectrozyme Xa, and measuring the increase in OD at 405 nm. Our studies show that a minimal amount of generated Xa (approximately 1nM) can be measured from ex-vivo thrombi. Static and shear samples generated the same amount of Xa, with the exception of blood subjected to 650 s(-1) shear. At 650 s(-1) shear rate, the amount of Xa generated reached a maximum of 4 nM at 5 min and then decreased to approximately 1 nM. Immunohistological stains and fluorescent images demonstrate the presence of cTF antigen at 650 s(-1) wall shear rate.

  14. Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

    PubMed

    Brzoska, Tomasz; Suzuki, Yuko; Sano, Hideto; Suzuki, Seiichirou; Tomczyk, Martyna; Tanaka, Hiroki; Urano, Tetsumei

    2017-04-03

    Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

  15. Supplementation of a Standardized Extract from Phyllanthus emblica Improves Cardiovascular Risk Factors and Platelet Aggregation in Overweight/Class-1 Obese Adults

    PubMed Central

    Khanna, Savita; Das, Amitava; Spieldenner, James; Rink, Cameron

    2015-01-01

    Abstract The objective of this study (clinicaltrials.gov NCT01858376) was to determine the effect of oral supplementation of a standardized extract of Phyllanthus emblica (CAPROS®) on cardiovascular disease (CVD) risk factors in overweight adult human subjects from the US population. Overweight/Class-1 obese (body–mass index: 25–35) adult subjects received 500 mg of CAPROS supplement b.i.d for 12 weeks. The study design included two baseline visits followed by 12 weeks of supplementation and then 2 weeks of washout. At all visits, peripheral venous blood was collected in sodium citrate tubes. Lipid profile measurements demonstrated a significant decrease in calculated low-density lipoprotein cholesterol and total cholesterol/high-density lipoprotein following 12 weeks of CAPROS supplementation when compared to averaged baseline visits. Circulatory high-sensitivity C reactive protein (hs-CRP) levels were significantly decreased after 12 weeks of supplementation. In addition, both ADP- and collagen-induced platelet aggregation was significantly downregulated following 12 weeks of supplementation. Overall, the study suggests that oral CAPROS supplementation may provide beneficial effects in overweight/Class-1 obese adults by lowering multiple global CVD risk factors. PMID:25756303

  16. Effect of Sclerovit on endothelial dysfunction, hemorheological parameters, platelet aggregation, plasma concentration of homocysteine and progression of atherosclerosis in patients with vascular diseases.

    PubMed

    Horvath, Beata; Szapary, Laszlo; Debreceni, Laszlo; Feher, Gergely; Kenyeres, Peter; Fulop, Adrienn; Battyani, Istvan; Toth, Kalman

    2009-01-01

    In our prospective study the effect of Sclerovit (0.8 mg folic acid, 20 mug vitamin B12,5 mg vitamin B6,100 mg vitamin E) on inflammatory markers, hemorheological parameters, platelet aggregation, von Willebrand factor activity as a marker of endothelium dysfunction, plasma lipids, plasma levels of folic acid, vitamin B12 and homocysteine (hcy), flow mediated vasodilatation (FMD) and thickness of carotis intima-media after 1 and 6 months of treatment in patients with vascular diseases (10 patients took 1 capsule, 10 patients 2 capsules of Sclerovit and 10 patients placebo) was determined.Plasma level of vitamin B12, folic acid and elongation index of red blood cells (RBC) increased significantly (p<0.05-0.001), hcy and triglyceride concentrations decreased significantly (p<0.05-0.001) in patients taking Sclerovit. HDL-cholesterol, RBC count, hematocrit, plasma and whole blood viscosity increased significantly (p<0.05-0.001) both in patients taking placebo or vitamins. Fibrinogen and CRP showed a significant (p<0.05-0.01) increase in patients on placebo, but did not change in patients on Sclerovit therapy. FMD showed a significant (p<0.05) amelioration in patients on 1 capsule of Sclerovit.Beside the favorable effects of Sclerovit on some of the measured parameters, the observed deterioration in hemorheological parameters can correlate with the contradictory results of large prospective studies with vitamins.

  17. Supplementation of a standardized extract from Phyllanthus emblica improves cardiovascular risk factors and platelet aggregation in overweight/class-1 obese adults.

    PubMed

    Khanna, Savita; Das, Amitava; Spieldenner, James; Rink, Cameron; Roy, Sashwati

    2015-04-01

    The objective of this study (clinicaltrials.gov NCT01858376) was to determine the effect of oral supplementation of a standardized extract of Phyllanthus emblica (CAPROS(®)) on cardiovascular disease (CVD) risk factors in overweight adult human subjects from the US population. Overweight/Class-1 obese (body-mass index: 25-35) adult subjects received 500 mg of CAPROS supplement b.i.d for 12 weeks. The study design included two baseline visits followed by 12 weeks of supplementation and then 2 weeks of washout. At all visits, peripheral venous blood was collected in sodium citrate tubes. Lipid profile measurements demonstrated a significant decrease in calculated low-density lipoprotein cholesterol and total cholesterol/high-density lipoprotein following 12 weeks of CAPROS supplementation when compared to averaged baseline visits. Circulatory high-sensitivity C reactive protein (hs-CRP) levels were significantly decreased after 12 weeks of supplementation. In addition, both ADP- and collagen-induced platelet aggregation was significantly downregulated following 12 weeks of supplementation. Overall, the study suggests that oral CAPROS supplementation may provide beneficial effects in overweight/Class-1 obese adults by lowering multiple global CVD risk factors.

  18. RhoA and Rac1 GTPases Differentially Regulate Agonist-Receptor Mediated Reactive Oxygen Species Generation in Platelets

    PubMed Central

    Akbar, Huzoor; Duan, Xin; Saleem, Saima; Davis, Ashley K.; Zheng, Yi

    2016-01-01

    Agonist induced generation of reactive oxygen species (ROS) by NADPH oxidases (NOX) enhances platelet aggregation and hence the risk of thrombosis. RhoA and Rac1 GTPases are involved in ROS generation by NOX in a variety of cells, but their roles in platelet ROS production remain unclear. In this study we used platelets from RhoA and Rac1 conditional knockout mice as well as human platelets treated with Rhosin and NSC23767, rationally designed small molecule inhibitors of RhoA and Rac GTPases, respectively, to better define the contributions of RhoA and Rac1 signaling to ROS generation and platelet activation. Treatment of platelets with Rhosin inhibited: (a) U46619 induced activation of RhoA; (b) phosphorylation of p47phox, a critical component of NOX; (c) U46619 or thrombin induced ROS generation; (d) phosphorylation of myosin light chain (MLC); (e) platelet shape change; (f) platelet spreading on immobilized fibrinogen; and (g) release of P-selectin, secretion of ATP and aggregation. Conditional deletion of RhoA or Rac1 gene inhibited thrombin induced ROS generation in platelets. Addition of Y27632, a RhoA inhibitor, NSC23766 or Phox-I, an inhibitor of Rac1-p67phox interaction, to human platelets blocked thrombin induced ROS generation. These data suggest that: (a) RhoA/ROCK/p47phox signaling axis promotes ROS production that, at least in part, contributes to platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC; and (b) RhoA and Rac1 differentially regulate ROS generation by inhibiting phosphorylation of p47phox and Rac1-p67phox interaction, respectively. PMID:27681226

  19. Methodology and outcomes of platelet aggregation testing in Australia, New Zealand and the Asia-Pacific region: results of a survey from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program.

    PubMed

    Duncan, E M; Bonar, R; Rodgers, S E; Favaloro, E J; Marsden, K

    2009-08-01

    Platelet aggregometry is widely used to investigate platelet function but its performance is poorly standardized between laboratories. The aim of this work was to document the platelet aggregation methods used in specialist laboratories enrolled in the Haematology Quality Assurance Program of the Royal College of Pathologists of Australasia. A questionnaire requesting many details of methodology was distributed and from the responses, we determined a consensus view. Consensus was defined here as >70% agreement among respondents in answer to a question and this was seen for a number of aspects of the preanalytical, analytical and interpretive phases. However, for many questions there was a wide variation in responses. Sixteen laboratories provided a breakdown of the types of abnormal results typically seen in a 12-month period. In these laboratories a total of 1400 patients were tested and 390 (27%) had abnormal platelet function. Although it was common to diagnose the cause as aspirin or an aspirin-like defect or a release/storage pool disorder, the range of experience was wide and other rare defects were reported. We conclude that whilst there are a number of points of agreement between laboratories in platelet function testing, standardization could be improved.

  20. Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis.

    PubMed

    Zhang, Yiwen; Zhang, Jian; Yan, Rong; Tian, Jingluan; Zhang, Yang; Zhang, Jie; Chen, Mengxing; Cui, Qingya; Zhao, Lili; Hu, Renping; Jiang, Miao; Li, Zhenyu; Ruan, Changgeng; He, Sudan; Dai, Kesheng

    2017-03-14

    Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3(-/-)) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3(-/-) bone marrow-derived cells had longer occlusion times than RIP3(-/-) mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3(-/-) platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

  1. Comparison between in vitro lipid peroxidation in fresh sheep platelets and peroxidative processes during sheep platelet ageing under storage at 4 degrees C.

    PubMed

    Martín-Valmaseda, E M; Sánchez-Yagüe, J; Rodríguez, M C; Gómez, F P; Llanillo, M

    1999-07-15

    Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.

  2. Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels in vivo and platelet aggregation in vitro.

    PubMed

    Nemmar, Abderrahim; Yuvaraju, Priya; Beegam, Sumaya; Ali, Badreldin H

    2015-07-01

    Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events in vivo and in vitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. In vitro, in whole blood samples collected from untreated mice (n = 3-5), betaine (0.01-1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen in vivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01-1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4-6). In conclusion, our data show that betaine protected against coagulation events in vivo and in vitro and decreased LPO in plasma.

  3. 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

  4. Isolation and identification of seven metabolites of a water-soluble platelet aggregation inhibitor in rat urine.

    PubMed

    Tanaka, M; Ishikawa, F; Hakusui, H

    1995-11-01

    1. Seven metabolites of 7-piperidino-1,2,3,4,5-tetrahydroimidazo[2,1- b]quinazolin-2-one dihydrochloride monohydrate (DN-9693) were isolated from rat urine by extraction with Amberlite XAD-2 and purification by silica gel and Sephadex LH-20 open-column chromatography and preparative high-performance liquid chromatography (hplc). The structure assignment of the metabolites was performed by field desorption mass spectrometry and 200-MHz Fourier transform nmr spectroscopic analysis and comparison with authentic standards when available. 2. DN-9693 underwent metabolism mainly at the piperidine ring to give the 4-hydroxypiperidine derivative (III) and 2-hydroxy-piperidine derivative, which is further metabolized to lactam (II) or delta-aminovaleric acid (V). The acyl side chain of V was shortened by beta-oxidation to form the 3-aminopropionic acid derivative (VII). V and/or VII underwent oxidative dealkylation to give the 7-amino derivative, which was conjugated with acetic acid to form the 7-acetylamino derivative (IV). DN-9693 also underwent hydrolysis of its lactam moiety to give VI. 3. The urinary excretion of III, V and VII was determined by liquid chromatography/electrochemistry (LC/EC) and V proved to be the major metabolite in rat urine. 4. A procedure is also presented for the identification of DN-9693 metabolites using LC/EC.

  5. Isolation of Phorbol Esters from Euphorbia grandicornis and Evaluation of Protein Kinase C- and Human Platelet-Activating Effects of Euphorbiaceae Diterpenes.

    PubMed

    Tsai, Ju-Ying; Rédei, Dóra; Forgo, Peter; Li, Yu; Vasas, Andrea; Hohmann, Judit; Wu, Chin-Chung

    2016-10-28

    Human platelets contain conventional (α and β) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives.

  6. [Initial selectivity of the antiplatelet covalent action of biogenic chloramines on platelet-rich plasma].

    PubMed

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

    2007-01-01

    To describe in full the peculiarities of the antiplatelet action of covalent inhibitors on platelet-rich plasma, we have proposed to take into account the initial selectivity that determines the elevated efficacy of inactivation of platelet molecular target (receptor). The quantitative index of initial selectivity is the ratio of rate constant of inactivation of the platelet molecular target to the rate constant of the chemical reaction of an inhibitor with reactive atomic groups in plasma proteins. For the important case of the domination of the inhibitor expenditure in the reaction with plasma proteins, a formula was derived which depicts the dependence of the share of inactivated targets on the concentration of the inhibitor introduced and reactive atomic groups contained in plasma. In the case of chloramine derivatives of amino acids, evidence was obtained indicating that the degree of inhibition of platelet aggregation measured by the turbidimetric method is equal to the square of the share of inactivated receptors. The index of initial selectivity can be evaluated by measuring the degree of inhibition of platelet aggregation and the operating concentration of the inhibitor. According to experimental evidence, the effects of a number of chloramine derivatives of amino acids (biochloramines) on aggregation of platelets stimulated by ADP show selectivity at the molecular target level, so that the index of initial selectivity is greater than 1. The mechanism of the selective action of the biochloramines having significant molecular masses (150-200 Da) probably consists in the inactivation of the molecular target via chemical modification of several reactive atomic groups in its different sites. One may suppose that the biochloramines with lower molecular masses (150-100 Da) exhibit a high anti-aggregatory capacity owing to another mechanism of initial selectivity, which involves the modification of highly sensitive sulfur-containing atomic groups.

  7. [Effects of selective microbial inhibitors on the microbial transformation of phosphorous in aggregates of highly weathered red soil with rice straw amendment].

    PubMed

    Ding, Long-jun; Xiao, He-ai; Wu, Jin-shui; Ge, Ti-da

    2010-07-01

    In order to further understand the mechanisms of microbial immobilization of phosphorous (P) in highly weathered red soil with organic amendment, an incubation test was conducted to investigate the roles of microbial functional groups in the transformation of P in 0.2-2 mm soil aggregates. Throughout the 90-day incubation period, amendment with rice straw induced a substantial increase in the amounts of microbial biomass C and P, Olsen-P, and organic P in the aggregates. Comparing with rice straw amendment alone, the amendment with rice straw plus fungal inhibitor actidione decreased the amount of microbial biomass C in the aggregates by 10.5%-31.8% in the first 30 days. Such a decrement was significantly larger than that (6.8%-11.6%) in the treatment amended with rice straw plus bacterial inhibitors tetracycline and streptomycin sulphate (P<0.01). After the first 30 days, the microbial biomass C remained constant. In the first 20 days, the amount of microbial biomass P in the aggregates was 10.0%-28.8% higher in the treatment amended with bacterial inhibitors than in the treatment amended with fungal inhibitor (P<0.01). All the results suggested that that both the fungal and the bacterial groups were involved in the microbial immobilization of P in the soil aggregates, and the fungal group played a relatively larger role.

  8. Endothelial cells microparticle-associated protein disulfide isomerase promotes platelet activation in metabolic syndrome

    PubMed Central

    Li, Yi-hui; Song, Dai-jun; Chen, Tong-shuai; Zhang, Wei; Zhong, Ming; Zhang, Yun; Xing, Yan-qiu; Wang, Zhi-hao

    2016-01-01

    Background Metabolic syndrome (MetS) is a common challenge in the world, and the platelet activation is enhanced in MetS patients. However, the fundamental mechanism that underlies platelet activation in MetS remains incompletely understood. Endothelial cells are damaged seriously in MetS patients, then they release more endothelial microparticles (EMPs). After all, whether the EMPs participate in platelet activation is still obscure. If they were, how did they work? Results We demonstrated that the levels of EMPs, PMPs (platelet derived microparticles) and microparticle-carried-PDI activity increased in MetS patients. IR endothelial cells released more EMPs, the EMP-PDI was more activated. EMPs can enhance the activation of CD62P, GPIIb/IIIa and platelet aggregation and this process can be partly inhibited by PDI inhibitor such as RL90 and rutin. Activated platelets stimulated by EMPs expressed more PDI on cytoplasm and released more PMPs. Materials and Methods We obtained plasma from 23 MetS patients and 8 normal healthy controls. First we built insulin resistance (IR) model of human umbilical vein endothelial cells (HUVECs), and then we separated EMPs from HUVECs culture medium and used these EMPs to stimulate platelets. Levels of microparticles, P-selectin(CD62P), Glycoprotein IIb/IIIa (GPIIb/IIIa) were detected by flow cytometry and levels of EMPs were detected by enzyme-linked immunosorbent assay (ELISA). The protein disulfide isomerase (PDI) activity was detected by insulin transhydrogenase assay. Platelet aggregation was assessed by turbidimetry. Conclusion EMPs can promote the activation of GPIIb/IIIa in platelets and platelet aggregation by the PDI which is carried on the surface of EMPs. PMID:27825126

  9. Ruthenium(II) polypyridyl complexes with hydrophobic ancillary ligand as Aβ aggregation inhibitors.

    PubMed

    Vyas, Nilima A; Ramteke, Shefali N; Kumbhar, Avinash S; Kulkarni, Prasad P; Jani, Vinod; Sonawane, Uddhavesh B; Joshi, Rajendra R; Joshi, Bimba; Erxleben, Andrea

    2016-10-04

    The synthesis, spectral and electrochemical characterization of the complexes of the type [Ru(NN)2(txbg)](2+) where NN is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), dipyrido [3,2-d:2',3f] quinoxaline (dpq) (3), and dipyrido[3,2-a:2',3'-c]phenazine (dppz) (4) which incorporate the tetra-xylene bipyridine glycoluril (txbg) as the ancillary ligand are described in detail. Crystal structures of ligand txbg and complex 2 were solved by single crystal X-ray diffraction. Thioflavin T (ThT) fluorescence and Transmission Electron Microscopy (TEM) results indicated that at micromolar concentration all complexes exhibit significant potential of Aβ aggregation inhibition, while the ligand txbg displayed weak activity towards Aβ aggregation. Complex 1 showed relatively low inhibition (70%) while complexes 2-4 inhibited nearly 100% Aβ aggregation after 240 h of incubation. The similar potential of complexes 2-4 and absence of any trend in their activity with the planarity of polypyridyl ligands suggests there is no marked effect of planarity of coligands on their inhibitory potential. Further studies on acetylcholinesterase (AChE) inhibition indicated very weak activity of these complexes against AChE. Detailed interactions of Aβ with both ligand and complex 2 have been studied by molecular modeling. Complex 2 showed interactions involving all three polypyridyl ligands with hydrophobic region of Aβ. Furthermore, the toxicity of these complexes towards human neuroblastoma cells was evaluated by MTT assay and except complex 4, the complexes displayed very low toxicity.

  10. Platelets: handle with care.

    PubMed

    Thomas, S

    2016-10-01

    Platelets are delicate cells that require careful handling between collection, preparation and transfusion. This review addresses practical questions relating to platelet concentration, resting time after collection, total time and number of periods without agitation and temperature. The bags in which platelets are stored are made from gas-permeable plastic to allow sufficient oxygen for the platelets to maintain aerobic respiration. Manufacturers have assigned limits for platelet content and concentration, and these must not be exceeded. There is no strong evidence for or against the resting of platelets post-collection and pre-agitation, but platelets should not be over-wrapped during this period as this compromises gas exchange; a short rest period of up to 1 h may allow the separation of minor aggregates. It is necessary to transport platelet concentrates (e.g. from manufacturing site to hospital), but these periods without gas exchange must be limited to avoid excessive damage to the platelets. Current data support a total of 24 h of transportation per component but with no individual period lasting more than 8 h. Platelets need to be stored at 20-24 °C based on evidence that colder storage leads to irreversible changes on the platelet membrane, resulting in phagocytosis of the platelets following transfusion. Storage at warmer temperatures may lead to an increase in bacterial risk. On the basis of this review, the UK Guidelines for Blood Transfusion Services have been updated to ensure that platelets are handled in the most appropriate way to ensure that efficacious components are provided for patients.

  11. Comparative evaluation of platelet-rich fibrin, mineral trioxide aggregate, and calcium hydroxide as pulpotomy agents in permanent molars with irreversible pulpitis: A randomized controlled trial

    PubMed Central

    Kumar, Varun; Juneja, Ruchi; Duhan, Jigyasa; Sangwan, Pankaj; Tewari, Sanjay

    2016-01-01

    Background: Pulpotomy has been proposed as an alternative for the management of irreversible pulpitis in permanent molars with closed apices. Aim: To compare the performances of calcium hydroxide (CH), mineral trioxide aggregate (MTA), and platelet-rich fibrin (PRF) as pulpotomy agents in mature permanent molars with irreversible pulpitis. Materials and Methods: Fifty-four permanent mandibular molars with carious exposure and symptoms of irreversible pulpitis were randomly allocated to three groups, and full pulpotomy was performed using CH, MTA, or PRF as pulpotomy agents. Pain intensity was recorded using numeric rating scale score at baseline, 24 h, 7 days, 6 months, and 1 year. Clinical and radiographic assessments were done at 6 months and 1 year. Statistical Analysis: Kruskal–Wallis test and Friedman test were used for intergroup and intragroup comparison of pain scores, respectively. The radiographic outcomes between the three study arms were compared using Chi-square test. Results: Clinical success rate was 94.4% at 7 days, which dropped to 85.4% at 12 months. All three agents were equally effective in providing pain relief at all the intervals tested, with no significant difference between them (P > 0.05 at all intervals). However, at 6 months and 12 months, 26.2% and 52.4% teeth depicted slight widening of periodontal ligament space. No significant difference was observed between the radiographic success rates observed with the three groups (P = 0.135 at 6 months, 0.717 at 12 months). Conclusion: Pulpotomy exhibited a high clinical success rate in mature molars with irreversible pulpitis and selection of biomaterial did not affect its outcome. PMID:27994420

  12. Isolation and characterization of two disintegrins inhibiting ADP-induced human platelet aggregation from the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake)

    SciTech Connect

    Sanchez, Elda E.; Galan, Jacob A.; Russell, William K.; Soto, Julio G.; Russell, David H.; Perez, John C. . E-mail: kfjcp00@tamuk.edu

    2006-04-01

    Disintegrins and disintegrin-like proteins are molecules found in the venom of four snake families (Atractaspididae, Elapidae, Viperidae, and Colubridae). The disintegrins are nonenzymatic proteins that inhibit cell-cell interactions, cell-matrix interactions, and signal transduction, and may have potential in the treatment of strokes, heart attacks, cancers, and osteoporosis. Prior to 1983, the venom of Crotalus scutulatus scutulatus (Mohave Rattlesnake) was known to be only neurotoxic; however, now there is evidence that these snakes can contain venom with: (1) neurotoxins; (2) hemorrhagins; and (3) both neurotoxins and hemorrhagins. In this study, two disintegrins, mojastin 1 and mojastin 2, from the venom of a Mohave rattlesnake collected in central Arizona (Pinal County), were isolated and characterized. The disintegrins in these venoms were identified by mass-analyzed laser desorption ionization/time-of-flight/time-of-flight (MALDI/TOF/TOF) mass spectrometry as having masses of 7.436 and 7.636 kDa. Their amino acid sequences are similar to crotratroxin, a disintegrin isolated from the venom of the western diamondback rattlesnake (C. atrox). The amino acid sequence of mojastin 1 was identical to the amino acid sequence of a disintegrin isolated from the venom of the Timber rattlesnake (C. horridus). The disintegrins from the Mohave rattlesnake venom were able to inhibit ADP-induced platelet aggregation in whole human blood both having IC{sub 5}s of 13.8 nM, but were not effective in inhibiting the binding of human urinary bladder carcinoma cells (T24) to fibronectin.

  13. Immunohistochemical Evaluation of Fibronectin and Tenascin Following Direct Pulp Capping with Mineral Trioxide Aggregate, Platelet-Rich Plasma and Propolis in Dogs’ Teeth

    PubMed Central

    Moradi, Saeed; Saghravanian, Nasrollah; Moushekhian, Siavash; Fatemi, Samar; Forghani, Maryam

    2015-01-01

    Introduction: The aim of the present study was to evaluate the expression of fibronectin (FN) and tenascin (TN) after direct pulp capping (DPC) in dogs’ teeth with either mineral trioxide aggregate (MTA), Propolis or Platelet-rich plasma (PRP), by means of immunohistochemistry. Methods and Materials: A total of 48 sound molars and premolars with mature apices from four dogs, were included. The teeth were randomly divided into 4 groups according to the material used for DPC: PRP, Propolis, MTA, and glass-ionomer (as the negative control group). Each group was divided into two 7-day and 30-day subgroups. The teeth were restored at the same session. The animals were sacrificed at the mentioned time intervals and the expression of FN and TN in each test group and between each time intervals was assessed with Wilcoxon and Mann-Whitney U tests, respectively. The Kruskal-Wallis test was used to compare FN and TN staining among the test groups. The significance level was set at 0.05. Results: The amount of FN in the MTA group in the 30-day interval was significantly higher than the 7-day interval; however, there were no significant differences among the other groups. The amount of TN in the MTA and Propolis groups in the 30-day interval was significantly higher than that in the 7-day interval; no recognizable difference was observed in the other groups. Moreover, the difference in expression of FN and TN in the 7-day interval was not significant in the experimental groups. Nevertheless, the difference was significant in the 30-day interval, with the highest and lowest expressions belonging to the MTA and glass-ionomer groups, respectively. Conclusion: Based on the results of the present animal study, MTA is still a better choice for direct pulp capping PMID:26213542

  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. 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.

  16. Importance of measurement of platelet reactivity to ADP in patients with coronary artery disease: an historical account.

    PubMed

    Tantry, Udaya S; Mahla, Elisabeth; Gesheff, Martin G; Gurbel, Paul A

    2013-11-01

    The pivotal roles of platelets in physiological hemostasis and pathological thrombosis at the site of plaque rupture are well established. The latter roles provide the fundamental basis for the most widely implemented pharmacologic management of coronary artery disease--dual antiplatelet therapy with aspirin to inhibit platelet thromboxane A2 generation, and a P2Y12 receptor inhibitor to prevent adenosine diphosphate (ADP)-induced platelet activation. Although suboptimal pharmacodynamic efficacy, also described as high on-treatment platelet reactivity to ADP, has been associated with greater risk for post-stenting ischemic event occurrence, enhanced responsiveness is associated with higher risk for bleeding in selected patients. In this review article, we aim to provide an historical account of the one and a half century long journey starting with the first description of platelets through the first report of ex vivo measurement of ADP-induced platelet aggregation, the first demonstration of an association between ADP-induced platelet aggregation and post-stenting ischemic event occurrence, and finally to the most recent description of a 'therapeutic window' concept for P2Y12 receptor inhibitor therapy.

  17. Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth.

    PubMed

    Pinar, Isaac P; Arthur, Jane F; Andrews, Robert K; Gardiner, Elizabeth E; Ryan, Kris; Carberry, Josie

    2015-01-01

    Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time.

  18. Methods to Determine the Lagrangian Shear Experienced by Platelets during Thrombus Growth

    PubMed Central

    Pinar, Isaac P.; Arthur, Jane F.; Andrews, Robert K.; Gardiner, Elizabeth E.; Ryan, Kris; Carberry, Josie

    2015-01-01

    Platelets can become activated in response to changes in flow-induced shear; however, the underlying molecular mechanisms are not clearly understood. Here we present new techniques for experimentally measuring the flow-induced shear rate experienced by platelets prior to adhering to a thrombus. We examined the dynamics of blood flow around experimentally grown thrombus geometries using a novel combination of experimental (ex vivo) and numerical (in silico) methodologies. Using a microcapillary system, platelet aggregate formation was analysed at elevated shear rates in the presence of coagulation inhibitors, where thrombus formation is predominantly platelet-dependent. These approaches permit the resolution and quantification of thrombus parameters at the scale of individual platelets (2 μm) in order to quantify real time thrombus development. Using our new techniques we can correlate the shear rate experienced by platelets with the extent of platelet adhesion and aggregation. The techniques presented offer the unique capacity to determine the flow properties for a temporally evolving thrombus field in real time. PMID:26660525

  19. Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors as novel approach to rapidly attenuate platelet-mediated thrombosis

    PubMed Central

    Gremmel, Thomas; Yanachkov, Ivan B.; Yanachkova, Milka I.; Wright, George E.; Wider, Joseph; Undyala, Vishnu V.R.; Michelson, Alan D.; Frelinger, Andrew L.; Przyklenk, Karin

    2015-01-01

    Objective Unlike currently approved adenosine diphosphate (ADP) receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet ADP receptor P2Y1, and may therefore be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409. Approach and Results We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited ADP- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, i.e., platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a non-significant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake. Conclusions Synergistic inhibition of both P2Y1 and P2Y12 ADP receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy. PMID:26743169

  20. AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets.

    PubMed

    Fleming, Ingrid; Schulz, Christian; Fichtlscherer, Birgit; Kemp, Bruce E; Fisslthaler, Beate; Busse, Rudi

    2003-11-01

    Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells. Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser(1177) but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca(2+) response to thrombin and was also prevented by N(omega) nitro-L-arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation. These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca(2+), by acti-vating PI3-K and AMPK which phosphorylates eNOS on Ser(1177).

  1. Thrombopoietin increases platelet adhesion under flow and decreases rolling.

    PubMed

    Van Os, Erim; Wu, Ya-Ping; Pouwels, Jos G; Ijsseldijk, Martin J W; Sixma, Jan J; Akkerman, Jan Willem N; De Groot, Philip G; Van Willigen, Gijsbert

    2003-05-01

    Thrombopoietin (TPO) is known to sensitize platelets to other agonists at 20 ng/ml, and above 100 ng/ml it is an independent activator of aggregation and secretion. In studies with a perfusion chamber, TPO, between 0.01 ng/ml and 1 ng/ml, increased platelet adhesion to surface-coated fibrinogen, fibronectin and von Willebrand Factor (VWF) but not to a collagen-coated surface. Increased adhesion was observed at shear rates of 300/s and 800/s in perfusions with whole blood as well as in suspensions of platelets and red blood cells reconstituted in plasma. The by the cyclooxygenase inhibitor, indomethacin, and the thromboxane A2-receptor blocker, SQ30741, abolished the stimulation by TPO. The effect of TPO was mimicked by a very low concentration (10 nmol/l) of the thromboxane TxA2 analogue, U46619. Real-time studies of platelet adhesion to a VWF-coated surface at a shear of 1000/s showed that about 20% of the platelets were in a rolling phase before they became firmly attached. TPO (1 ng/ml) pretreatment reduced this number to < 5%, an effect again abolished by indomethacin. Thus, TPO potentiates the direct and firm attachment of platelets to surface-coated ligands for alphaIIbbeta3, possibly by increasing the ligand affinity of the integrin.

  2. Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies

    PubMed Central

    Fuentes, Eduardo; Pereira, Jaime; Alarcón, Marcelo; Valenzuela, Claudio; Pérez, Pablo; Astudillo, Luis; Palomo, Iván

    2013-01-01

    The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5′-monophosphate) by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P < 0.05). Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1 μm2 (P < 0.001). After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P < 0.001). Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study) and thrombosis formation in vivo (murine model) were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids) to processed foods. PMID:24159349

  3. Protective Mechanisms of S. lycopersicum Aqueous Fraction (Nucleosides and Flavonoids) on Platelet Activation and Thrombus Formation: In Vitro, Ex Vivo and In Vivo Studies.

    PubMed

    Fuentes, Eduardo; Pereira, Jaime; Alarcón, Marcelo; Valenzuela, Claudio; Pérez, Pablo; Astudillo, Luis; Palomo, Iván

    2013-01-01

    The purpose of this research was to investigate mechanisms of antiplatelet action of bioactive principle from S. lycopersicum. Aqueous fraction had a high content of nucleosides (adenosine, guanosine, and adenosine 5'-monophosphate) by HPLC analysis. Also aqueous fraction presented flavonoids content. Aqueous fraction inhibited platelet activation by 15 ± 6% (P < 0.05). Fully spread of human platelets on collagen in the presence of aqueous fraction was inhibited from 15 ± 1 to 9 ± 1  μ m(2) (P < 0.001). After incubation of whole blood with aqueous fraction, the platelet coverage was inhibited by 55 ± 12% (P < 0.001). Platelet ATP secretion and aggregation were significantly inhibited by the aqueous fraction. At the same concentrations that aqueous fraction inhibits platelet aggregation, levels of sCD40L significantly decreased and the intraplatelet cAMP levels increased. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the effect of aqueous fraction toward ADP-induced platelet aggregation and intraplatelet level of cAMP. Platelet aggregation ex vivo (human study) and thrombosis formation in vivo (murine model) were inhibited by aqueous fraction. Finally, aqueous fraction may be used as a functional ingredient adding antiplatelet activities (nucleosides and flavonoids) to processed foods.

  4. The Kinetics of Circulating Monocyte Subsets and Monocyte-Platelet Aggregates in the Acute Phase of ST-Elevation Myocardial Infarction

    PubMed Central

    Zhou, Xin; Liu, Xin-Lin; Ji, Wen-Jie; Liu, Jun-Xiang; Guo, Zhao-Zeng; Ren, Dong; Ma, Yong-Qiang; Zeng, Shan; Xu, Zhong-Wei; Li, Hong-Xia; Wang, Peizhong Peter; Zhang, Zhuoli; Li, Yu-Ming; Benefield, Brandon C.; Zawada, Adam M.; Thorp, Edward B.; Lee, Daniel C.; Heine, Gunnar H.

    2016-01-01

    Abstract In experimental myocardial infarction (MI), a rise in cell counts of circulating monocyte subsets contributes to impaired myocardial healing and to atherosclerotic plaque destabilization. In humans, the prognostic role of monocyte subsets in patients suffering ST-elevation MI (STEMI) is still unclear. In the present study, we aimed to determine the kinetics of the 3 monocyte subsets (classical CD14++CD16–, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes), as well as the subset-specific monocyte–platelet aggregates (MPA), in acute STEMI followed by primary percutaneous coronary intervention (PCI), and their relationships with cardiovascular outcomes during a 2-year follow-up. Monocyte subsets and MPA were measured in 100 STEMI patients receiving primary PCI on days 1, 2, 3, 5, and 7 of symptom onset, which were compared with 60 stable coronary heart disease patients and 35 healthy volunteers. From day 1 to day 7, significant increases in the counts of CD14++CD16+ monocytes and CD14++CD16+ MPA were observed, with peak levels on day 2. During a median follow-up of 2.0 years, 28 first cardiovascular events (defined as cardiovascular death, nonfatal ischemic stroke, recurrent MI, need for emergency or repeat revascularization, and rehospitalization for heart failure) were recorded. After adjustment for confounders, CD14++CD16+ monocytosis (day 1 [HR: 3.428; 95% CI: 1.597–7.358; P = 0.002], day 2 [HR: 4.835; 95% CI: 1.106–21.13; P = 0.04], day 3 [HR: 2.734; 95% CI: 1.138–6.564; P = 0.02], and day 7 [HR: 2.647; 95% CI: 1.196–5.861; P = 0.02]), as well as increased levels of CD14++CD16+ MPA measured on all time points (days 1, 2, 3, 5, and 7), had predictive values for adverse cardiovascular events. In conclusion, our data show the expansion of the CD14++CD16+ monocyte subset during acute phase of STEMI has predictive values for 2-year adverse cardiovascular outcomes in patients treated with primary PCI. Future studies

  5. The Kinetics of Circulating Monocyte Subsets and Monocyte-Platelet Aggregates in the Acute Phase of ST-Elevation Myocardial Infarction: Associations with 2-Year Cardiovascular Events.

    PubMed

    Zhou, Xin; Liu, Xin-Lin; Ji, Wen-Jie; Liu, Jun-Xiang; Guo, Zhao-Zeng; Ren, Dong; Ma, Yong-Qiang; Zeng, Shan; Xu, Zhong-Wei; Li, Hong-Xia; Wang, Peizhong Peter; Zhang, Zhuoli; Li, Yu-Ming; Benefield, Brandon C; Zawada, Adam M; Thorp, Edward B; Lee, Daniel C; Heine, Gunnar H

    2016-05-01

    In experimental myocardial infarction (MI), a rise in cell counts of circulating monocyte subsets contributes to impaired myocardial healing and to atherosclerotic plaque destabilization. In humans, the prognostic role of monocyte subsets in patients suffering ST-elevation MI (STEMI) is still unclear. In the present study, we aimed to determine the kinetics of the 3 monocyte subsets (classical CD14++CD16-, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes), as well as the subset-specific monocyte-platelet aggregates (MPA), in acute STEMI followed by primary percutaneous coronary intervention (PCI), and their relationships with cardiovascular outcomes during a 2-year follow-up.Monocyte subsets and MPA were measured in 100 STEMI patients receiving primary PCI on days 1, 2, 3, 5, and 7 of symptom onset, which were compared with 60 stable coronary heart disease patients and 35 healthy volunteers. From day 1 to day 7, significant increases in the counts of CD14++CD16+ monocytes and CD14++CD16+ MPA were observed, with peak levels on day 2. During a median follow-up of 2.0 years, 28 first cardiovascular events (defined as cardiovascular death, nonfatal ischemic stroke, recurrent MI, need for emergency or repeat revascularization, and rehospitalization for heart failure) were recorded. After adjustment for confounders, CD14++CD16+ monocytosis (day 1 [HR: 3.428; 95% CI: 1.597-7.358; P = 0.002], day 2 [HR: 4.835; 95% CI: 1.106-21.13; P = 0.04], day 3 [HR: 2.734; 95% CI: 1.138-6.564; P = 0.02], and day 7 [HR: 2.647; 95% CI: 1.196-5.861; P = 0.02]), as well as increased levels of CD14++CD16+ MPA measured on all time points (days 1, 2, 3, 5, and 7), had predictive values for adverse cardiovascular events.In conclusion, our data show the expansion of the CD14++CD16+ monocyte subset during acute phase of STEMI has predictive values for 2-year adverse cardiovascular outcomes in patients treated with primary PCI. Future studies will be warranted to

  6. 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

  7. JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets.

    PubMed

    Naik, Meghna U; Stalker, Timothy J; Brass, Lawrence F; Naik, Ulhas P

    2012-04-05

    Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)β(3). Once platelet activation has occurred, integrin α(IIb)β(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A-deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

  8. [Selective serotonin reuptake inhibitor and gastrointestinal hemorrhage].

    PubMed

    Yamamoto, Takatsugu; Abe, Koichiro; Kuyama, Yasushi

    2013-04-01

    Selective serotonin reuptake inhibitors (SSRI) are widely used antidepressants characterized by less-frequent adverse effects compared with classical anti-depressive agents. On the other hand, SSRI can cause hemorrhagic events more due to impaired platelet aggregation induced by a depletion of serotonin in the peripheral platelet. Epidemiological studies have indicated that patients taking SSRI are predisposed to gastrointestinal hemorrhage, especially in case that nonsteroidal anti-inflammatory drugs are prescribed concomitantly. Here we describe a risk of the gastrointestinal hemorrhage in patients taking SSRI.

  9. Biomimetic design of platelet adhesion inhibitors to block integrin α2β1-collagen interactions: I. Construction of an affinity binding model.

    PubMed

    Zhang, Lin; Sun, Yan

    2014-04-29

    Platelet adhesion on a collagen surface through integrin α2β1 has been proven to be significant for the formation of arterial thrombus. However, the molecular determinants mediating the integrin-collagen complex remain unclear. In the present study, the dynamics of integrin-collagen binding and molecular interactions were investigated using molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) analysis. Hydrophobic interaction is identified as the major driving force for the formation of the integrin-collagen complex. On the basis of the MD simulation and MM-PBSA results, an affinity binding model (ABM) of integrin for collagen is constructed; it is composed of five residues, including Y157, N154, S155, R288, and L220. The ABM has been proven to capture the major binding motif contributing 84.8% of the total binding free energy. On the basis of the ABM, we expect to establish a biomimetic design strategy of platelet adhesion inhibitors, which would be beneficial for the development of potent peptide-based drugs for thrombotic diseases.

  10. How do the full-generation poly(amido)amine (PAMAM) dendrimers activate blood platelets? Activation of circulating platelets and formation of "fibrinogen aggregates" in the presence of polycations.

    PubMed

    Watala, Cezary; Karolczak, Kamil; Kassassir, Hassan; Talar, Marcin; Przygodzki, Tomasz; Maczynska, Katarzyna; Labieniec-Watala, Magdalena

    2016-04-30

    Direct use of poly(amido)amine (PAMAM) dendrimers as drugs may be limited, due to uncertain (cyto)toxicity. Peripheral blood components, which constitute the first line of a contact with administered pharmaceuticals, may become vastly affected by PAMAM dendrimers. The aim of this study was to explore how PAMAMs' polycationicity might affect blood platelet activation and reactivity, and thus trigger various haemostatic events. We monitored blood platelet reactivity in rats with experimental diabetes upon a long-term administration of the unmodified PAMAM dendrimers. In parallel, the effects on blood flow in a systemic circulation was recorded intravitally in mice administered with PAMAM G2, G3 or G4. Compounding was the in vitro approach to monitor the impact of PAMAM dendrimers on blood platelet activation and reactivity and on selected haemostatic and protein conformation parameters. We demonstrated the activating effects of polycations on blood platelets. Some diversity of the revealed outcomes considerably depended on the used approach and the particular technique employed to monitor blood platelet function. We discovered undesirable impact of plain PAMAM dendrimers on primary haemostasis and their prothrombotic influence. We emphasize the need of a more profound verifying of all the promising findings collected for PAMAMs with the use of well-designed in vivo preclinical studies.

  11. Epinephrine--via activation of p38-MAPK--abolishes the effect of aspirin on platelet deposition to collagen.

    PubMed

    Mustonen, P; van Willigen, G; Lassila, R

    2001-12-15

    The mechanism by which epinephrine enhances experimental thrombosis in the presence of aspirin is poorly understood. In this study, we set to explore, in aspirinised platelet-rich plasma (PRP), the effect of epinephrine (100 nmol/l) on platelet deposition to immobilised collagen and the subsequent involvement of several intracellular pathways. Under these experimental conditions, which allow platelet aggregation on top of the collagen-adherent platelets, epinephrine increased platelet deposition by 55-86%. This enhancement could be specifically prohibited by the alpha(2A)-adrenoceptor antagonist, atipamezole, the p38 mitogen-activated protein kinase (p38MAPK) inhibitor SB203580, and the cytosolic phospholipase A(2) (cPLA(2)) inhibitor, mepacrine. The effect of epinephrine coincided with increased phosphorylation of p38MAPK and cPLA(2) and with arachidonic acid (AA) release from platelet membrane. We conclude that epinephrine enhanced platelet deposition on collagen in aspirinised PRP via a mechanism dependent on both free AA in platelet cytosol (released by cPLA(2)) and p38MAPK.

  12. A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease

    PubMed Central

    Myöhänen, TT; Hannula, MJ; Van Elzen, R; Gerard, M; Van Der Veken, P; García-Horsman, JA; Baekelandt, V; Männistö, PT; Lambeir, AM

    2012-01-01

    BACKGROUND AND PURPOSE The aggregation of α-synuclein is connected to the pathology of Parkinson's disease and prolyl oligopeptidase (PREP) accelerates the aggregation of α-synuclein in vitro. The aim of this study was to investigate the effects of a PREP inhibitor, KYP-2047, on α-synuclein aggregation in cell lines overexpressing wild-type or A30P/A53T mutant human α-syn and in the brains of two A30P α-synuclein transgenic mouse strains. EXPERIMENTAL APPROACH Cells were exposed to oxidative stress and then incubated with the PREP inhibitor during or after the stress. Wild-type or transgenic mice were treated for 5 days with KYP-2047 (2 × 3 mg·kg−1 a day). Besides immunohistochemistry and thioflavin S staining, soluble and insoluble α-synuclein protein levels were measured by Western blot. α-synuclein mRNA levels were quantified by PCR. The colocalization of PREP and α-synuclein,and the effect of KYP-2047 on cell viability were also investigated. KEY RESULTS In cell lines, oxidative stress induced a robust aggregation of α-synuclein,and low concentrations of KYP-2047 significantly reduced the number of cells with α-synuclein inclusions while abolishing the colocalization of α-synuclein and PREP. KYP-2047 significantly reduced the amount of aggregated α-synuclein,and it had beneficial effects on cell viability. In the transgenic mice, a 5-day treatment with the PREP inhibitor reduced the amount of α-synuclein immunoreactivity and soluble α-synuclein protein in the brain. CONCLUSIONS AND IMPLICATIONS The results suggest that the PREP may play a role in brain accumulation and aggregation of α-synuclein, while KYP-2047 seems to effectively prevent these processes. PMID:22233220

  13. 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.

  14. Expression, purification, and analysis of three recombinant ECD disintegrins (r-colombistatins) from P-III class snake venom metalloproteinases affecting platelet aggregation and SK-MEL-28 cell adhesion

    PubMed Central

    Suntravat, Montamas; Helmke, Thomas J.; Atphaisit, Chairat; Cuevas, Esteban; Lucena, Sara E.; Uzcátegui, Nestor L.; Sánchez, Elda E.; Rodriguez-Acosta, Alexis

    2016-01-01

    Crotalid venoms are rich sources of components that affect the hemostatic system. Snake venom metalloproteinases are zinc-dependent enzymes responsible for hemorrhage that also interfere with hemostasis. The disintegrin domain is a part of snake venom metalloproteinases, which involves the binding of integrin receptors. Integrins play an essential role in cancer survival and invasion, and they have been major targets for drug development and design. Both native and recombinant disintegrins have been widely investigated for their anti-cancer activities in biological systems as well as in vitro and in vivo systems. Here, three new cDNAs encoding ECD disintegrin-like domains of metalloproteinase precursor sequences obtained from a Venezuelan mapanare (Bothrops colombiensis) venom gland cDNA library have been cloned. Three different N- and C-terminal truncated ECD disintegrin-like domains of metalloproteinases named colombistatins 2, 3, and 4 were amplified by PCR, cloned into a pGEX-4T-1 vector, expressed in Escherichia coli BL21, and tested for inhibition of platelet aggregation and inhibition of adhesion of human skin melanoma (SK-Mel-28) cancer cell lines on collagen I. Purified recombinant colombistatins 2, 3, and 4 were able to inhibit ristocetin- and collagen-induced platelet aggregation. r-Colombistatins 2 showed the most potent inhibiting SK-Mel-28 cancer cells adhesion to collagen. These results suggest that colombistatins may have utility in the development of therapeutic tools in the treatment of melanoma cancers and also thrombotic diseases. PMID:27641750

  15. Influence of red algal sulfated polysaccharides on blood coagulation and platelets activation in vitro.

    PubMed

    Sokolova, Ekaterina V; Byankina, Anna O; Kalitnik, Alexandra A; Kim, Yong H; Bogdanovich, Larisa N; Solov'eva, Tamara F; Yermak, Irina M

    2014-05-01

    The influence of sulfated polysaccharides (λ-, κ-, and κ/β-carrageenan and porphyran) - on platelet activation was studied. Carrageenans were much weaker inhibitors of a coagulation process than heparin, while porphyran had not that effect. Results of the aPTT and PT assays suppose that carrageenans affected mostly intrinsic pathway of coagulation, while their effect on the extrinsic pathway is extremely low (λ and κ/β) or absent (κ, LMW derivative of κ-carrageenan). λ-Carrageenan was the most potent anticoagulant agent in TT, aPTT, PT, and anti-factor Xa activity. This sample was also the strongest inhibitor of collagen-induced platelet aggregation in PRP. Generally, the correlation of anticoagulant and antithrombotic action in PRP is preserved for carrageenans but not for heparin. Carrageenans and porphyran affected platelet adhesion to collagen by influencing glycoprotein VI. Low molecular weight κ-carrageenan had a similar effect on platelet adhesion mediated with both major collagen receptors: integrin α2 β1 and glycoprotein VI as native polysaccharide had. Carrageenans resulted in activation of platelets under platelet adhesion mediated by integrin αIIb β3 with less degree than heparin. The least sulfated κ/β-carrageenan that possessed an inhibiting effect on thrombin- and collagen-induced aggregation of washed platelets and on the PT test but it had no significant effect on TT was the weakest promoter of integrin αIIb β3 mediated platelet activation. In summary, our study showed that the polysaccharide action was complex, since it depended on its molecular mass, sulfation degree, and monosaccharide contents (3,6-anhydrogalactose).

  16. Identification of membrane proteins mediating the interaction of human platelets

    PubMed Central

    Phillips, D; Jennings, L; Edwards, H

    1980-01-01

    Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that

  17. Platelet abnormalities in adults with severe pulmonary arterial hypertension related to congenital heart defects (Eisenmenger syndrome).

    PubMed

    Remková, Anna; Šimková, Iveta; Valkovičová, Tatiana; Kaldarárová, Monika

    2016-12-01

    Patients with severe pulmonary arterial hypertension suffer from life-threatening thrombotic and bleeding complications. The aim of this study was to compare selected platelet, endothelial, and coagulation parameters in healthy volunteers and patients with severe pulmonary arterial hypertension because of congenital heart defects. The study included healthy volunteers (n = 50) and patients with cyanotic congenital heart defects classified as Eisenmenger syndrome (n = 41). We investigated platelet count, mean platelet volume, and platelet aggregation - spontaneous and induced by various concentrations of five agonists. Von Willebrand factor (vWF), fibrinogen, factor VIII and XII, plasminogen activator inhibitor, antithrombin, D-dimer, and antiphospholipid antibodies were also investigated. We found a decreased platelet count [190 (147-225) vs. 248 (205-295) 10 l, P < 0.0001], higher mean platelet volume [10.9 (10.1-12.0) vs. 10.2 (9.4-10.4) fl, P < 0.0001], and significantly decreased platelet aggregation (induced by five agonists, in various concentrations) in patients with Eisenmenger syndrome compared with controls. These changes were accompanied by an increase of plasma vWF antigen [141.6 (108.9-179.1) vs. 117.4 (9.2-140.7) IU/dl, P = 0.022] and serum anti-β2-glycoprotein [2.07 (0.71-3.41) vs. 0.47 (0.18-0.99) U/ml, P < 0.0001]. Eisenmenger syndrome is accompanied by platelet abnormalities. Thrombocytopenia with increased platelet size is probably due to a higher platelet turnover associated with platelet activation. Impaired platelet aggregation can reflect specific platelet behaviour in patients with Eisenmenger syndrome. These changes can be related both to bleeding and to thrombotic events. A higher vWF antigen may be a consequence of endothelial damage in Eisenmenger syndrome, but the cause for an increase of anti-β2-glycoprotein is unknown.

  18. Salivary Antigen-5/CAP Family Members Are Cu2+-dependent Antioxidant Enzymes That Scavenge O2⨪ and Inhibit Collagen-induced Platelet Aggregation and Neutrophil Oxidative Burst*

    PubMed Central

    Assumpção, Teresa C. F.; Ma, Dongying; Schwarz, Alexandra; Reiter, Karine; Santana, Jaime M.; Andersen, John F.; Ribeiro, José M. C.; Nardone, Glenn; Yu, Lee L.; Francischetti, Ivo M. B.

    2013-01-01

    The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism. PMID:23564450

  19. Transcriptomic Analysis of the Ion Channelome of Human Platelets and Megakaryocytic Cell Lines

    PubMed Central

    Wright, Joy R.; Amisten, Stefan; Goodall, Alison H.; Mahaut-Smith, Martyn P.

    2016-01-01

    Summary Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288–11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These “channelome” datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte. PMID:27277069

  20. Human platelets repurposed as vehicles for in vivo imaging of myeloma xenotransplants.

    PubMed

    Dai, Lu; Gu, Ning; Chen, Bao-An; Marriott, Gerard

    2016-04-19

    Human platelets were identified in tumors by Trousseau in 1865, although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers. In this study we exploit and enhance platelet interactions in tumor microenvironments by introducing tumor-targeting and imaging functions. The first step in repurposing human platelets as vehicles for tumor-targeting was to inhibit platelet-aggregation by cytoplasmic-loading of kabiramide (KabC), a potent inhibitor of actin polymerization and membrane protrusion. KabC-Platelets can accumulate high levels of other membrane-permeable cytoxins and probes, including epidoxorubicin, carboxyfluorescein di-ester and chlorin-e6. Finally, mild reaction conditions were developed to couple tumor-targeting proteins and antibodies to KabC-platelets. Fluorescence microscopy studies showed KabC-platelets, surface-coupled with transferrin and Cy5, bind specifically to RPMI8226 and K562 cells, both of which over-express the transferrin receptor. Repurposed platelets circulate for upto 9-days a feature that increases their chance of interacting with target cells. KabC-platelets, surface-coupled with transferrin and Cy7, or chlorin-e6, and injected in immuno-compromised mice were shown to accumulate specifically in sub-cutaneous and intra-cranial myeloma xenotransplants. The high-contrast, in vivo fluorescence images recorded from repurposed platelets within early-stage myeloma is a consequence in part of their large size (φ~2µm), which allows them to transport 100 to 1000-times more targeting-protein and probe molecules respectively. Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help stage tumor environments for an immunotherapy, or with combinations of therapeutic antibodies and therapeutic agents to target and treat cardiovascular and neurologic diseases.

  1. Human platelets repurposed as vehicles for in vivo imaging of myeloma xenotransplants

    PubMed Central

    Dai, Lu; Gu, Ning; Chen, Bao-An; Marriott, Gerard

    2016-01-01

    Human platelets were identified in tumors by Trousseau in 1865, although their roles in tumor microenvironments have only recently attracted the attention of cancer researchers. In this study we exploit and enhance platelet interactions in tumor microenvironments by introducing tumor-targeting and imaging functions. The first step in repurposing human platelets as vehicles for tumor-targeting was to inhibit platelet-aggregation by cytoplasmic-loading of kabiramide (KabC), a potent inhibitor of actin polymerization and membrane protrusion. KabC-Platelets can accumulate high levels of other membrane-permeable cytoxins and probes, including epidoxorubicin, carboxyfluorescein di-ester and chlorin-e6. Finally, mild reaction conditions were developed to couple tumor-targeting proteins and antibodies to KabC-platelets. Fluorescence microscopy studies showed KabC-platelets, surface-coupled with transferrin and Cy5, bind specifically to RPMI8226 and K562 cells, both of which over-express the transferrin receptor. Repurposed platelets circulate for upto 9-days a feature that increases their chance of interacting with target cells. KabC-platelets, surface-coupled with transferrin and Cy7, or chlorin-e6, and injected in immuno-compromised mice were shown to accumulate specifically in sub-cutaneous and intra-cranial myeloma xenotransplants. The high-contrast, in vivo fluorescence images recorded from repurposed platelets within early-stage myeloma is a consequence in part of their large size (φ∼2μm), which allows them to transport 100 to 1000-times more targeting-protein and probe molecules respectively. Human platelets can be configured with a plurality of therapeutic and targeting antibodies to help stage tumor environments for an immunotherapy, or with combinations of therapeutic antibodies and therapeutic agents to target and treat cardiovascular and neurologic diseases. PMID:27049725

  2. 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

  3. Platelets in Critical Illness.

    PubMed

    Levi, Marcel

    2016-04-01

    In patients with critical illness, thrombocytopenia is a frequent laboratory abnormality. However frequent this may occur, a low platelet count is not an epiphenomenon, but a marker with further significance. It is always important to assess the proper cause for thrombocytopenia in critically ill patients because different underlying disorders may precipitate different diagnostic and therapeutic management strategies. Platelets are part of the first-line defense of the body against bleeding; hence, thrombocytopenia may increase the risk of hemorrhage. In case of systemic inflammatory syndromes, such as the response to sepsis, disseminated intravascular platelet activation may occur. This will contribute to microvascular failure and thereby play a role in the development of organ dysfunction. Platelets are circulating blood cells that will normally not interact with the intact vessel wall but that may swiftly respond to endothelial disruption (which is often part of the pathogenesis of critical illness) by adhering to subendothelial structures, followed by interaction with each other, thereby forming a platelet aggregate. The activated platelet (phospholipid) membrane may form a suitable surface on which further coagulation activation may occur. A low platelet count is a strong and independent predictor of an adverse outcome in critically ill patients, thereby facilitating a simple and practically risk assessment in these patients and potentially guiding the use of complex or expensive treatment strategies.

  4. Optimization of potent DFG-in inhibitors of platelet derived growth factor receptorβ (PDGF-Rβ) guided by water thermodynamics.

    PubMed

    Horbert, Rebecca; Pinchuk, Boris; Johannes, Eugen; Schlosser, Joachim; Schmidt, Dorian; Cappel, Daniel; Totzke, Frank; Schächtele, Christoph; Peifer, Christian

    2015-01-08

    In this study we report on the hit optimization of substituted 3,5-diaryl-pyrazin-2(1H)-ones toward potent and effective platelet-derived growth factor receptor (PDGF-R) β-inhibitors. Originally, the 3,5-diaryl-pyrazin-2-one core was derived from the marine sponge alkaloid family of hamacanthins. In our first series compound 2 was discovered as a promising hit showing strong activity against PDGF-Rβ in the kinase assay (IC50 = 0.5 μM). Furthermore, 2 was shown to be selective for PDGF-Rβ in a panel of 24 therapeutically relevant protein kinases. Molecular modeling studies on a PDGF-Rβ homology model using prediction of water thermodynamics suggested an optimization strategy for the 3,5-diaryl-pyrazin-2-ones as DFG-in binders by using a phenolic OH function to replace a structural water molecule in the ATP binding site. Indeed, we identified compound 38 as a highly potent inhibitor with an IC50 value of 0.02 μM in a PDGF-Rβ enzymatic assay also showing activity against PDGF-R dependent cancer cells.

  5. 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.

  6. 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.

  7. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor.

    PubMed Central

    MacPhee, C H; Moores, K E; Boyd, H F; Dhanak, D; Ife, R J; Leach, C A; Leake, D S; Milliner, K J; Patterson, R A; Suckling, K E; Tew, D G; Hickey, D M

    1999-01-01

    A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine. PMID:10024526

  8. MALT1-ubiquitination triggers non-genomic NF-κB/IKK signaling upon platelet activation.

    PubMed

    Karim, Zubair A; Vemana, Hari Priya; Khasawneh, Fadi T

    2015-01-01

    We have recently shown that IKK complex plays an important non-genomic role in platelet function, i.e., regulates SNARE machinery-dependent membrane fusion. In this connection, it is well known that MALT1, whose activity is modulated by proteasome, plays an important role in the regulation of IKK complex. Therefore, the present studies investigated the mechanism by which IKK signaling is regulated in the context of the platelet proteasome. It was found that platelets express a functional proteasome, and form CARMA/MALT1/Bcl10 (CBM) complex when activated. Using a pharmacological inhibitor, the proteasome was found to regulate platelet function (aggregation, integrin activation, secretion, phosphatidylserine exposure and changes in intracellular calcium). It was also found to regulate thrombogenesis and physiologic hemostasis. We also observed, upon platelet activation, that MALT1 is ubiquitinated, and this coincides with the activation of the IKK/NF-κB-signaling pathway. Finally, we observed that the proteasome inhibitor blocks CBM complex formation and the interaction of IKKγ and MALT1; abrogates SNARE formation, and the association of MALT1 with TAK1 and TAB2, which are upstream of the CBM complex. Thus, our data demonstrate that MALT1 ubiquitination is critical for the engagement of CBM and IKK complexes, thereby directing platelet signals to the NF-κB pathway.

  9. 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.

  10. 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 b