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Sample records for platelet aggregation inhibitor

  1. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

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

  2. 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.

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

    PubMed

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

    2011-10-01

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

  4. Inhibitors of ex vivo aggregation of human platelets induced by decompression, during reduced barometric pressure.

    PubMed

    Murayama, M; Kumaroo, K K

    1986-05-15

    It has been shown experimentally ex vivo that human platelet aggregation is induced by decompression (reduced pressure) produced by various means, i.e., reduced barometric pressure, reduced hydrostatic pressure, and reduced hydrodynamic pressure due to Bernoulli's principle. We report here that the spontaneous platelet aggregation induced by reduced barometric pressure (253 torr for three hours) is inhibited by 1:10(7) diluted Japanese herbal plant oil (JHP) and also by two of its major constituents, menthone and menthol with the median inhibitory concentration (IC50) in the millimolar range. These drugs gave essentially similar results when collagen and ADP were used as aggregating agents. Inhibitor concentrations were determined by microscopic examination of platelets in wet preparations when the aggregating stimulus was reduced pressure and by optical aggregometry when collagen and ADP were the aggregating agents. Potential usefulness of these compounds in the prevention of decompression syndrome (DCS) and acute mountain sickness (AMS) are discussed.

  5. Mechanism for release of arachidonic acid during guinea pig platelet aggregation: a role for the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.

    1986-01-01

    The mechanism of the release of arachidonic acid from phospholipids after the stimulation of guinea pig platelets with collagen, thrombin and platelet activating factor (PAF) was studied. RHC 80267, a diacylglycerol lipase inhibitor, and indomethacin, a cyclooxygenase inhibitor, were used. Various in vitro assays for enzymes involved in arachidonic acid release and metabolism were conducted. Platelet aggregation and simultaneous release of ADP from platelets were monitored using a Chrono-log Lumiaggregometer. Platelets were labeled with (/sup 14/C)arachidonic acid to facilitate sensitive determination of small changes in platelet phospholipids during platelet aggregation. In the present investigation it is shown that collagen, thrombin and PAF increased phospholipase C activity. It was also discovered that cyclooxygenase products were responsible for further stimulation (a positive feed-back) of phospholipase C activity, while diacylglycerol provided a negative feed-back control over receptor-stimulated phospholipase C activity and inhibited ADP release. The guinea pig platelet is an ideal model to study phospholipase C-diacylglycerol lipase pathway for the release of arachidonic acid from platelet phospholipids because it does not have any phospholipase A/sub 2/ activity. It was observed that cyclooxygenase products were responsible for collagen-induced guinea pig platelet aggregation. Indomethacin completely inhibited collagen-induced platelet aggregation, was less effective against thrombin, and had no effect on PAF-induced platelet aggregation. On the other hand, RHC 80267 was a powerful inhibitor of aggregation and ADP release induced by all three of these potent aggregating agents.

  6. Echistatin. A potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus.

    PubMed

    Gan, Z R; Gould, R J; Jacobs, J W; Friedman, P A; Polokoff, M A

    1988-12-25

    A 49-residue protein, echistatin, which inhibits platelet aggregation, was purified from the venom of the saw-scaled viper Echis carinatus. The purification procedure included gel filtration on Sephadex G-50, cation-exchange chromatography on Mono S, and C18 reverse-phase high pressure liquid chromatography. The purified protein was homogeneous as judged by polyacrylamide gel electrophoresis, isoelectric focusing, reverse-phase high pressure liquid chromatography, and NH2-terminal sequence analysis. Echistatin is a single-chain polypeptide with a molecular weight of 5400 and a native isoelectric point of 8.3. The most abundant amino acid, cysteine, accounts for 8 of the 49 residues in the protein. A 10-residue segment of echistatin shows 90% identity to a portion of the sequence of trigramin, a platelet aggregation inhibitor from the green tree viper Trimereserus gramineus (Huang, T.-F., Holt, J. C., Lukasiewicz, H., and Niewiarowski, S. (1987) J. Biol. Chem. 262, 16157-16163). Echistatin contains the sequence arginine-glycine-aspartic acid, which is common to proteins which bind to the glycoprotein IIb/IIIa complex. It also contains the sequence proline-arginine-asparagine-proline, which is found in the A alpha chain of human fibrinogen at position 267-270. The purified protein inhibits fibrinogen-dependent platelet aggregation initiated by ADP with an IC50 of 3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of echistatin abolished its inhibitory activity. PMID:3198653

  7. Taurine and platelet aggregation

    SciTech Connect

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

    1986-03-01

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

  8. Nutmeg oil: identification and quantitation of its most active constituents as inhibitors of platelet aggregation.

    PubMed

    Janssens, J; Laekeman, G M; Pieters, L A; Totte, J; Herman, A G; Vlietinck, A J

    1990-05-01

    Three distilled or commercially available nutmeg oils were analysed and their chemical composition compared with their capacity to inhibit platelet aggregation in vitro. It could be clearly shown that eugenol and isoeugenol play the major role in the detected activity of nutmeg. Medicinally, it appears that nutmeg oil and nutmeg powder can be replaced by eugenol and/or isoeugenol. PMID:2115612

  9. 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

  10. 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

  11. Identification and characterization of a collagen-induced platelet aggregation inhibitor, triplatin, from salivary glands of the assassin bug, Triatoma infestans.

    PubMed

    Morita, Akihiro; Isawa, Haruhiko; Orito, Yuki; Iwanaga, Shiroh; Chinzei, Yasuo; Yuda, Masao

    2006-07-01

    To facilitate feeding, certain hematophagous invertebrates possess inhibitors of collagen-induced platelet aggregation in their saliva. However, their mechanisms of action have not been fully elucidated. Here, we describe two major salivary proteins, triplatin-1 and -2, from the assassin bug, Triatoma infestans, which inhibited platelet aggregation induced by collagen but not by other agents including ADP, arachidonic acid, U46619 and thrombin. Furthermore, these triplatins also inhibited platelet aggregation induced by collagen-related peptide, a specific agonist of the major collagen-signaling receptor glycoprotein (GP)VI. Moreover, triplatin-1 inhibited Fc receptor gamma-chain phosphorylation induced by collagen, which is the first step of GPVI-mediated signaling. These results strongly suggest that triplatins target GPVI and inhibit signal transduction necessary for platelet activation by collagen. This is the first report on the mechanism of action of collagen-induced platelet aggregation inhibitors from hematophagus invertebrates. PMID:16759235

  12. Ticagrelor--a new platelet aggregation inhibitor in patients with acute coronary syndromes. An improvement of other inhibitors?

    PubMed

    Kowalczyk, Mariusz; Banach, Maciej; Mikhailidis, Dimitri P; Hannam, Simon; Rysz, Jacek

    2009-12-01

    Antiplatelet agents play an essential role in the treatment of acute coronary syndrome (ACS). Thienopyridines are a class of drugs that function via inhibition of the adenosine diphosphate (ADP) P2Y12 platelet receptors. Currently, clopidogrel, a second generation thienopyridine, is the main drug of choice and the combination of aspirin and clopidogrel is administered orally for the treatment of ACS. Clopidogrel, is a pro-drug that needs to be metabolized in the liver and intestines to form active metabolites. Prasugrel, a third generation thienopyridine, was approved for use in Europe in February 2009, and is currently available in the United Kingdom. All thienopyridines however, have pharmacological limitations that lead to a search for more effective non-thienopyridine P2Y12 inhibitors. Promising results have been reported with ticagrelor, an oral first reversible, direct-acting inhibitor of the P2Y12 receptor. Ticagrelor is the first oral P2Y12 receptor binding antagonist that does not require metabolic activation. Furthermore, ticagrelor has at last 1 active metabolite, which has very similar pharmacokinetics to the parent compound. Therefore, ticagrelor has more rapid onset and more pronounced platelet inhibition than other antiplatelet agents. The safety and efficacy of ticagrelor compared with clopidogrel in ACS patient has been recently evaluated by the PLATelet inhibition and patient Outcomes (PLATO) trial. Ticagrelor compared with clopidogrel had a significantly greater reduction in the death rate from vascular causes, myocardial infarction, or stroke without major bleeding. There was however, an increase in non-procedure related bleeding, dyspnoea and ventricular pauses in the first week of treatment. Further studies on new antiplatelet agents are needed to establish a new "gold standard" antiplatelet therapy. PMID:19946242

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

    PubMed

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

    2011-04-01

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

  14. MK-886, an inhibitor of the 5-lipoxygenase-activating protein, inhibits cyclooxygenase-1 activity and suppresses platelet aggregation.

    PubMed

    Koeberle, Andreas; Siemoneit, Ulf; Northoff, Hinnak; Hofmann, Bettina; Schneider, Gisbert; Werz, Oliver

    2009-04-17

    MK-886, an inhibitor of the 5-lipoxygenase-activating protein (FLAP), potently suppresses leukotriene biosynthesis in intact cells and is frequently used to define a role of the 5-lipoxygenase (EC 1.13.11.34) pathway in cellular or animal models of inflammation, allergy, cancer, and cardiovascular disease. Here we show that MK-886 also interferes with the activities of cyclooxygenases (COX, EC 1.14.99.1). MK-886 inhibited isolated COX-1 (IC(50)=8 microM) and blocked the formation of the COX-1-derived products 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid (12-HHT) and thromboxane B(2) in washed human platelets in response to collagen as well as from exogenous arachidonic acid (IC(50)=13-15 microM). Isolated COX-2 was less affected (IC(50)=58 microM), and in A549 cells, MK-886 (33 microM) failed to suppress COX-2-dependent 6-keto-prostaglandin (PG)F(1alpha) formation. The distinct susceptibility of MK-886 towards COX-1 and -2 is apparent in automated molecular docking studies that indicate a preferred binding of MK-886 to COX-1 into the active site. MK-886 (10 microM) inhibited COX-1-mediated platelet aggregation induced by collagen or arachidonic acid whereas thrombin- or U-46619-induced (COX-independent) aggregation was not affected. Since leukotrienes and prostaglandins share (patho)physiological properties in the development and regulation of carcinogenesis, inflammation, and vascular functions, caution should be used when interpreting data where MK-886 is used as tool to determine the involvement of FLAP and/or the 5-lipoxygenase pathway in respective experimental models.

  15. Cyclosporine A enhances platelet aggregation.

    PubMed

    Grace, A A; Barradas, M A; Mikhailidis, D P; Jeremy, J Y; Moorhead, J F; Sweny, P; Dandona, P

    1987-12-01

    In view of the reported increase in thromboembolic episodes following cyclosporine A (CyA) therapy, the effect of this drug on platelet aggregation and thromboxane A2 release was investigated. The addition of CyA, at therapeutic concentrations to platelet rich plasma from normal subjects in vitro was found to increase aggregation in response to adrenaline, collagen and ADP. Ingestion of CyA by healthy volunteers was also associated with enhanced platelet aggregation. The CyA-mediated enhancement of aggregation was further enhanced by the addition in vitro of therapeutic concentrations of heparin. Platelets from renal allograft recipients treated with CyA also showed hyperaggregability and increased thromboxane A2 release, which were most marked at "peak" plasma CyA concentration and less so at "trough" concentrations. Platelet hyperaggregability in renal allograft patients on long-term CyA therapy tended to revert towards normal following the replacement of CyA with azathioprine. Hypertensive patients with renal allografts on nifedipine therapy had normal platelet function and thromboxane release in spite of CyA therapy. These observations suggest that CyA-mediated platelet activation may contribute to the pathogenesis of the thromboembolic phenomena associated with the use of this drug. The increased release of thromboxane A2 (a vasoconstrictor) may also play a role in mediating CyA-related nephrotoxicity.

  16. 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.

  17. Discovery and preliminary SAR of 5-arylidene-2,2-dimethyl-1,3-dioxane- 4,6-diones as platelet aggregation inhibitors.

    PubMed

    El Maatougui, Abdelaziz; Azuaje, Jhonny; Coelho, Alberto; Cano, Ernesto; Yañez, Matilde; López, Carmen; Yaziji, Vicente; Carbajales, Carlos; Sotelo, Eddy

    2012-08-01

    We herein document the discovery of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones as a novel family of platelet aggregation inhibitors. The preliminary optimization study enabled us to establish the most salient features of the structure-activity relationships in this series as well as to identify novel derivatives that are upto 60 times more potent than the hit structure 1 and slightly superior to the reference drug Milrinone. PMID:22272691

  18. Discovery and preliminary SAR of 5-arylidene-2,2-dimethyl-1,3-dioxane- 4,6-diones as platelet aggregation inhibitors.

    PubMed

    El Maatougui, Abdelaziz; Azuaje, Jhonny; Coelho, Alberto; Cano, Ernesto; Yañez, Matilde; López, Carmen; Yaziji, Vicente; Carbajales, Carlos; Sotelo, Eddy

    2012-08-01

    We herein document the discovery of 5-arylidene-2,2-dimethyl-1,3-dioxane-4,6-diones as a novel family of platelet aggregation inhibitors. The preliminary optimization study enabled us to establish the most salient features of the structure-activity relationships in this series as well as to identify novel derivatives that are upto 60 times more potent than the hit structure 1 and slightly superior to the reference drug Milrinone.

  19. The saliva proteome of the blood-feeding insect Triatoma infestans is rich in platelet-aggregation inhibitors

    NASA Astrophysics Data System (ADS)

    Charneau, Sébastien; Junqueira, Magno; Costa, Camila M.; Pires, Daniele L.; Fernandes, Ellen S.; Bussacos, Ana C.; Sousa, Marcelo V.; Ricart, Carlos André O.; Shevchenko, Andrej; Teixeira, Antonio R. L.

    2007-12-01

    The saliva of the bloodsucking bug Triatoma infestans vector of Chagas disease contains an anti-hemostatic molecular cocktail that prevents coagulation, vasoconstriction and platelet aggregation in a vertebrate prey. In order to characterize T. infestans saliva proteome, we separated the secreted saliva by two-dimensional gel electrophoresis (2-DE). More than 200 salivary proteins were detected on the 2-DE map, mainly in the alkaline region. By nanoLC-MS/MS analysis using a LTQ-Orbitrap equipment followed by a combination of conventional and sequence-similarity searches, we identified 58 main protein spots. Most of such proteins possess potential blood-feeding associated functions, particularly anti-platelet aggregation proteins belonging to lipocalin and apyrase families. The saliva protein composition indicates a highly specific molecular mechanism of early response to platelet aggregation. This first proteome analysis of the T. infestans secreted saliva provides a basis for a better understanding of this fluid protein composition highly directed to counterpart hemostasis of the prey, thus promoting the bug's blood-feeding.

  20. Chemical synthesis of echistatin, a potent inhibitor of platelet aggregation from Echis carinatus: synthesis and biological activity of selected analogs.

    PubMed

    Garsky, V M; Lumma, P K; Freidinger, R M; Pitzenberger, S M; Randall, W C; Veber, D F; Gould, R J; Friedman, P A

    1989-06-01

    Echistatin, a polypeptide from the venom of the saw-scaled viper, Echis carinatus, containing 49 amino acids and 4 cystine bridges was synthesized by solid-phase methodology in 4% yield. In the final step, air oxidation of the octahydroderivative was found to be optimal at pH 8. The synthetic product was shown to be physically and biologically indistinguishable from native material. It inhibits fibrinogen-dependent platelet aggregation stimulated by ADP with IC50 = 3.3 x 10(-8) M and also prevents aggregation initiated by thrombin, epinephrine, collagen, or platelet-activating factor. Reduction of purified synthetic echistatin to octahydroechistatin with dithiothreitol followed by air oxidation regenerated homogeneous echistatin in quantitative yield. This highly specific refolding strongly suggests that the linear sequence of octahydroechistatin contains all of the information that is required for the proper folding of the peptide. The sequence Arg24-Gly-Asp of echistatin occurs also in adhesive glycoproteins that bind to the platelet fibrinogen receptor--a heterodimeric complex composed of glycoproteins IIb and IIIa. In an effort to evaluate the role of this putative binding site we have synthesized analogs of echistatin with substitution of Arg-24. Replacement with ornithine-24 (Orn-24) resulted in an analog having a platelet aggregation inhibitory activity with IC50 = 1.05 x 10(-7) M. Substitution with Ala-24 gave IC50 = 6.1 x 10(-7) M. The inhibitory activity of the corresponding short sequence analogs Arg-Gly-Asp-Phe (IC50 = 6 x 10(-6) M), Orn-Gly-Asp-Phe (IC50 = 1.3 x 10(-4) M), and Ala-Gly-Asp-Phe (IC50 = 5.0 x 10(-4) M) was also determined. These results suggest that arginine plays a more important role in the binding of the tetrapeptide than in that of echistatin. PMID:2726764

  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. New Acyloxy Nitroso Compounds with Improved Water Solubility and Nitroxyl (HNO) Release Kinetics and Inhibitors of Platelet Aggregation

    PubMed Central

    Mohamed, Heba A. H.; Abdel-Aziz, Mohamed; Abuo-Rahma, Gamal El-Din A. A.; King, S. Bruce.

    2015-01-01

    New acyloxy nitroso compounds, 4-nitrosotetrahydro-2H-pyran-4-yl 2,2,2-trichloroacetate and 4-nitrosotetrahydro-2H-pyran-4-yl 2,2-dichloropropanoate were prepared. These compounds release HNO under neutral conditions with half-lives between 50 and 120 minutes, identifying these HNO donors as kinetically intermediate to the much slower acetate derivative and the faster trifluoroacetic acid derivative. These compounds or HNO-derived from these compounds react with thiols, including glutathione, thiol-containing enzymes and heme-containing proteins in a similar fashion to other acyloxy nitroso compounds. HNO released from these acyloxy nitroso compounds inhibits activated platelet aggregation. These acyloxy nitroso compounds augment the range of release for this group of HNO donors and should be valuable tools in the further study of HNO biology. PMID:26228501

  3. Lonomia obliqua venomous secretion induces human platelet adhesion and aggregation.

    PubMed

    Berger, Markus; Reck, José; Terra, Renata M S; Beys da Silva, Walter O; Santi, Lucélia; Pinto, Antônio F M; Vainstein, Marilene H; Termignoni, Carlos; Guimarães, Jorge A

    2010-10-01

    The caterpillar Lonomia obliqua is a venomous animal that causes numerous accidents, especially in southern Brazil, where it is considered a public health problem. The clinical manifestations include several haemostatic disturbances that lead to a hemorrhagic syndrome. Considering that platelets play a central role in hemostasis, in this work we investigate the effects of L. obliqua venomous secretion upon blood platelets responses in vitro. Results obtained shows that L. obliqua venom directly induces aggregation and ATP secretion in human washed platelets in a dose-dependent manner. Electron microscopy studies clearly showed that the venomous bristle extract was also able to produce direct platelets shape change and adhesion as well as activation and formation of platelet aggregates. Differently from other enzyme inhibitors, the venom-induced platelet aggregation was significatively inhibited by p-bromophenacyl bromide, a specific inhibitor of phospholipases A2. Additional experiments with different pharmacological antagonists indicate that the aggregation response triggered by the venom active components occurs through a calcium-dependent mechanism involving arachidonic acid metabolite(s) of the cyclooxygenase pathway and activation of phosphodiesterase 3A, an enzyme that leads to the consumption of intracellular cAMP content. It was additionally found that L. obliqua-induced platelet aggregation was independent of ADP release. Altogether, these findings are in line with the need for a better understanding of the complex hemorrhagic syndrome resulting from the envenomation caused by L. obliqua caterpillars, and can also give new insights into the management of its clinical profile.

  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. 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.

  6. Acidic-store depletion is required for human platelet aggregation.

    PubMed

    Amor, Nidhal Ben; Zbidi, Hanene; Bouaziz, Aicha; Jardin, Isaac; Isaac, Jardin; Hernández-Cruz, Juan M; Salido, Ginés M; Rosado, Juan A; Bartegi, Aghleb

    2009-10-01

    Platelet stimulation with thrombin induces an elevation in cytoplasmic free Ca(2+) concentration ([Ca(2+)]c) due to Ca(2+) release from intracellular stores and entry from the extracellular medium. Two different intracellular Ca(2+) stores have been described in human platelets: the dense tubular system and the lysosomal-like acidic stores. In the present study, we investigated the contribution of the acidic stores in thrombin-induced platelet aggregation. We have found that platelet aggregation induced by thrombin is reduced in a Ca(2+)-free medium. Discharge of the acidic Ca(2+) stores by treatment with the sarcoendoplasmic Ca(2+)-ATPase (SERCA)3 selective inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone reduced thrombin-evoked platelet aggregation. In the presence of 2,5-di-(tert-butyl)-1,4-hydroquinone, platelet aggregation induced by the protease-activated receptor (PAR)-1 and PAR-4 agonist peptides, SFLLRN and AYPGKF, respectively, was significantly reduced. In cells with depleted acidic stores, activation of GPIb-IX-V by thrombin resulted in reduced or no platelet aggregation in a medium containing 1 mmol/l Caor in a Ca(2+)-free medium, respectively. This finding suggests that Ca(2+) accumulation in the acidic Ca(2+) compartments is required for platelet aggregation induced by activation of the G-coupled PAR-1 and PAR-4 thrombin receptors and, by the occupation of the leucine-rich glycoprotein GPIb-IX-V and provide evidence supporting a functional role of the lysosomal-like acidic Ca(2+) stores in human platelets. PMID:19587585

  7. Anger expression correlates with platelet aggregation.

    PubMed

    Wenneberg, S R; Schneider, R H; Walton, K G; MacLean, C R; Levitsky, D K; Mandarino, J V; Waziri, R; Wallace, R K

    1997-01-01

    Potential relationships between increased platelet aggregability and such psychological characteristics as hostility and anger were investigated as part of a larger intervention study investigating the potential efficacy of stress-reduction treatments. Participants performed 6-minute mental arithmetic tests under time pressure. Blood was sampled during the first minute of the task and whole blood platelet aggregation was measured in an aggregometer, using collagen and ADP. To assess anger and hostility, the authors used Spielberger's State-Trait Anger and Anger Expression scales together with the Cook-Medley Hostility Scale. The authors found positive correlations between collagen-induced platelet aggregation and outwardly expressed anger, as measured by the Anger Expression Scale. The findings suggested that modes of anger expression may be associated with increased platelet aggregation. If confirmed by future studies, this finding could provide a mechanism for the putative connection between anger/hostility and coronary heart disease.

  8. 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.

  9. Increasing platelet aggregability after venepuncture is platelet, not plasma derived.

    PubMed

    Terres, W; Becker, B F; Kratzer, M A; Gerlach, E

    1986-05-15

    The time course of ADP induced aggregation of human platelets was determined in aliquots of stored platelet rich plasma 3.5, 10, 30 and 100 minutes after venepuncture. The maximal rate of aggregation was found to increase throughout this entire period, even though pH (7.4), CO2 (7 volume per cent) and temperature (35 degrees C) of the samples were kept constant. The mean acceleration (+/- SEM) between 3.5 and 100 minutes was 41.7 +/- 6.9 per cent (n = 67) at an ADP-concentration of 1 mumol/l and 18.3 +/- 6.2 per cent (n = 23) at 2 mumol/l ADP. The effect did not result from changes of any platelet regulatory factors putatively present alone in the plasma. Acceleration of aggregability was only found when the platelets themselves underwent storage, but not when freshly prepared plasma was given to prestored platelets. The change in aggregability was not diminished after inhibition of platelet cyclooxygenase by oral administration of acetylsalicylic acid. PMID:3715816

  10. An efficient and safe process for the preparation of ticagrelor, a platelet aggregation inhibitor via resin-NO2 catalyzed formation of triazole ring.

    PubMed

    Shinde, Gorakshanath B; Mahale, Pravin K; Padaki, Santhosh A; Niphade, Navnath C; Toche, Raghunath B; Mathad, Vijayavitthal T

    2015-01-01

    An efficient, safe and improved process for the preparation of ticagrelor 1, a platelet aggregation inhibitor is described. Synthesis comprises the condensation of pyrimidine amine derivative 14 with cyclopentyl derivative 13 in ethylene glycol followed by construction of triazole compound 16 by diazotization of the obtained intermediate 15 with a green and safer reagent "Resin-NO2" in water and acetonitrile mixture. Condensation of 16 with cyclopropylamine derivative 10 followed by deprotection of compound 12 with hydrochloric acid in dichloromethane (DCM) furnished ticagrelor 1 with an overall yield of 65 % and purity of 99.78 % by HPLC. Each reaction stage was optimized independently to establish the scalable and plant friendly process. An efficient and a safe process for key intermediate 14 which involve nitration reaction has also been developed. Safety parameters were established by understanding the thermal events of the reaction by DSC analysis.Graphical abstractSynthesis of ticagrelor via resin-NO2 catalysed formation of triazole ring. PMID:26389018

  11. Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

    PubMed Central

    Liu, L X; Weller, P F

    1992-01-01

    The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids. Images PMID:1313445

  12. 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.

  13. Inhibiting platelets aggregation could aggravate the acute infection caused by Staphylococcus aureus.

    PubMed

    Zhang, Xin; Liu, Yu; Gao, Yaping; Dong, Jie; Mu, Chunhua; Lu, Qiang; Shao, Ningsheng; Yang, Guang

    2011-01-01

    Several fibrinogen binding proteins (Fibs) play important roles in the pathogenesis of Staphylococcus aureus (S. aureus). Most Fibs can promote the aggregation of platelets during infection, but the extracellular fibrinogen-binding protein (Efb) is an exception. It is reported that Efb can specifically bind fibrinogen and inhibit the aggregation of platelet with its N terminal. However, the biological significance of platelet aggregation inhibition in the infection caused by S. aureus is unclear until now. Here, we demonstrated that the persistence and aggregation of platelets were important for killing S. aureus in whole blood. It was found that the N terminal of Efb (EfbN) and platelets inhibitors could increase the survival of S. aureus in whole blood. The study in vivo also showed that EfbN and platelets inhibitors could reduce the killing of S. aureus and increase the lethality rate of S. aureus in the acute infection mouse model.

  14. Hemostatic Mechanisms, Independent of Platelet Aggregation, Arrest Gastric Mucosal Bleeding

    NASA Astrophysics Data System (ADS)

    Whittle, Brendan J. R.; Kauffman, Gordon L.; Moncada, Salvador

    1986-08-01

    Platelet adhesion, aggregation, and subsequent plug formation play a major role in the control of cutaneous and vascular hemostasis. Little is known, however, about the hemostatic processes in gastric mucosal tissue. A method for evaluating bleeding from a standard incision in the gastric mucosa of the rat, rabbit, and dog has therefore been developed. By using pharmacological agents that interfere with platelet aggregation and blood coagulation, the mechanism of gastric hemostasis has been compared to that in the vasculature, using the rat mesenteric artery. Intravenous infusion of prostacyclin (0.5 μ g\\cdot kg-1\\cdot min-1), which inhibits platelet aggregation directly, or administration of the thromboxane synthase inhibitor 1-benzylimidazole (50 mg\\cdot kg-1) significantly prolonged bleeding in the mesenteric artery yet failed to alter gastric mucosal bleeding. In contrast, a low dose of heparin (100 units\\cdot kg-1), which interferes with the clotting process, had no effect on mesenteric bleeding but substantially prolonged bleeding from the gastric mucosa. These findings suggest that, unlike in the skin or vasculature, platelet aggregation plays a minimal role in the initial hemostatic events in the gastric mucosa and that the arrest of gastric hemorrhage is brought about largely by processes primarily involving the coagulation system.

  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. Platelet aggregation monitoring with a newly developed quartz crystal microbalance system as an alternative to optical platelet aggregometry.

    PubMed

    Sinn, Stefan; Müller, Lothar; Drechsel, Hartmut; Wandel, Michael; Northoff, Hinnak; Ziemer, Gerhard; Wendel, Hans P; Gehring, Frank K

    2010-11-01

    The objective of this study was to establish a new test system for the monitoring of platelet aggregation during extracorporeal circulation (ECC) procedures. Even though extensive progress has been made in improving the haemocompatibility of extracorporeal circulation devices, activation of blood coagulation, blood platelets and inflammatory responses are still undesired outcomes of cardiopulmonary bypass. This study deals with an approach towards a platelet aggregation measuring system using a newly developed quartz crystal microbalance (QCM) system. Since QCM is a rarely used technique in the field of blood analytics, the challenge was to transfer the well established methods of aggregometry to the new test system. In a QCM system, either bare gold or fibrinogen-coated sensors were incubated with ADP or arachidonic acid (AA) stimulated platelet rich plasma. For negative controls the GPIIb/IIIa inhibitory antibody abciximab (Reopro®) was used as an inhibitor of platelet aggregation. During incubation, the frequency shifts of the sensors were recorded. The results gained from the QCM system were compared to results gained by optical platelet aggregometry (born aggregometry). For additional visualization of platelet adhesion to the sensor surfaces, fluorescent microscopy and scanning electron microscopy were used. The QCM sensor was able to detect platelet aggregation in both uncoated and fibrinogen coated sensors. The measuring curves of aggregation measurements and controls were clearly distinguishable from each other in terms of frequency shifts and kinetics. For aggregation measurements and inhibited controls the therapeutic diagnosis of platelet function is identical between aggregometer and QCM data. In future, QCM based measuring devices may become an alternative to established point of care methods for rapid bedside testing of platelet aggregation.

  18. [A method for studying intravascular platelet aggregation in vitro].

    PubMed

    Ikonnikova, E I; Chernousova, L A; Moshkina, I R

    1999-06-01

    A simple available method for evaluating intravascular platelet aggregation is proposed. It consists in graphic recording of disaggregation of platelet-rich citrate plasma, which indicates the degree of intravascular aggregation. Intravascular aggregation is notably increased in coronary patients and negligible in normal subjects. The method may be used for the diagnosis of diseases with a high thrombogenic risk.

  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. Purification and characterization of a platelet aggregation inhibitor acidic phospholipase A2 from Indian saw-scaled viper (Echis carinatus) venom.

    PubMed

    Kemparaju, K; Krishnakanth, T P; Veerabasappa Gowda, T

    1999-12-01

    An acidic phospholipase A2 (EC-I-PLA2) has been purified from the Indian saw-scaled viper (Echis carinatus) venom through a combination of column chromatography and electrophoresis. EC-I-PLA2 has a molecular weight of 16000 by SDS-PAGE. It was focussed between pH 4.2 and 4.8 by isoelectro focussing. EC-I-PLA2 was non-lethal to mice and devoid of neurotoxicity, myotoxicity, anticoagulant activity and cytotoxicity. It induced mild oedema in the foot pads of mice. The purified PLA2 inhibited ADP, collagen and epinephrine induced human platelet aggregation and the inhibition was both dose and time dependent. PMID:10519645

  1. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

  2. A critical role for the regulation of Syk from agglutination to aggregation in human platelets.

    PubMed

    Shih, Chun-Ho; Chiang, Tin-Bin; Wang, Wen-Jeng

    2014-01-10

    Agglucetin, a tetrameric glycoprotein (GP) Ibα agonist from Formosan Agkistrodon acutus venom, has been characterized as an agglutination inducer in human washed platelets (WPs). In platelet-rich plasma (PRP), agglucetin dramatically elicits a biphasic response of agglutination and subsequent aggregation. For clarifying the intracellular signaling events from agglutination to aggregation in human platelets, we examined the essential signaling molecules involved through the detection of protein tyrosine phosphorylation (PTP). In WPs, an anti-GPIbα monoclonal antibody (mAb) AP1, but not a Src kinase inhibitor PP1, completely inhibited agglucetin-induced agglutination. However, PP1 but not AP1 had a potent suppression on platelet aggregation by a GPVI activator convulxin. The PTP analyses showed agglucetin alone can cause a weak pattern involving sequential phosphorylation of Lyn/Fyn, Syk, SLP-76 and phospholipase Cγ2 (PLCγ2). Furthermore, a Syk-selective kinase inhibitor, piceatannol, significantly suppressed the aggregating response in agglucetin-activated PRP. Analyzed by flow cytometry, the binding capacity of fluorophore-conjugated PAC-1, a mAb recognizing activated integrin αIIbβ3, was shown to increase in agglucetin-stimulated platelets. Again, piceatannol but not PP1 had a concentration-dependent suppression on agglucetin-induced αIIbβ3 exposure. Moreover, the formation of signalosome, including Syk, SLP-76, VAV, adhesion and degranulation promoting adapter protein (ADAP) and PLCγ2, are required for platelet aggregation in agglucetin/fibrinogen-activated platelets. In addition, GPIbα-ligation via agglucetin can substantially promote the interactions between αIIbβ3 and fibrinogen. Therefore, the signal pathway of Lyn/Fyn/Syk/SLP-76/ADAP/VAV/PLCγ2/PKC is sufficient to trigger platelet aggregation in agglucetin/fibrinogen-pretreated platelets. Importantly, Syk may function as a major regulator for the response from GPIbα-initiated agglutination to

  3. 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. PMID:19172515

  4. [THE INFLUENCE OF HYDROGEN SULFIDE ON COLLAGEN-INDUCED AGGREGATION OF HUMAN PLATELETS].

    PubMed

    Petrova, I V; Trubacheva, O A; Mangataeva, O S; Suslova, T E; Kovalev, I V; Gusakova, S V

    2015-10-01

    Study the impact of hydrogen sulfide on collagen-induced platelet aggregation from healthy donors and patients with type 2 diabetes. In healthy individuals, in contrast to patients with type 2 diabetes, NaHS significantly inhibited platelet aggregation. Activators of cAMP signaling (forskolin and phosphodiesterase inhibitor) significantly reduced platelet aggregation in both groups of examinees. NO-synthase inhibitors increased platelet aggregation in healthy volunteers, but not in patients with type 2 diabetes. The presence of H2S donor did not alter the extent of platelet aggregation at high concentrations of cAMP or decreased production of nitric oxide. It is assumed that the antiplatelet effect of H2S is not associated with the effect on the signal system, mediated cAMP or nitric oxide. Change H2S-dependent regulation of platelet aggregation in patients with type 2 diabetes is caused by disorders have been reported with this disease: the increase of intracellular calcium ion concentration, oxidative damage to proteins, hyperhomocysteinemia, glycosylation of key proteins involved in this process.

  5. [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. PMID:24989289

  6. [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.

  7. 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.

  8. 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

  9. 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

  10. Inhibition of platelet aggregation and reduced formation of thromboxane and lipoxygenase products in platelets by oil of cloves.

    PubMed

    Srivastava, K C; Justesen, U

    1987-09-01

    Oil of cloves (OC) was found to be a potent inhibitor of platelet aggregation induced by arachidonic acid (AA), collagen and epinephrine; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by OC seems to be mediated through a reduced formation of thromboxane as indicated by the following experimental evidence. (i) OC inhibited TxB2 formation in intact as well as lysed platelet preparations from added arachidonate, and (ii) it inhibited the formation of TxB2 from AA-labelled platelets after activation with Ca2+-ionophore A23187. The formation of lipoxygenase derived products was dependent on the concentration of OC used; at its lower concentration their amounts increased but this was found to be reversed at higher concentrations. At all concentrations thromboxane was decreased with a concomitant increase in unused AA. PMID:3118394

  11. 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. PMID:19880317

  12. 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

  13. Effect of selective inhibition of the p38 MAP kinase pathway on platelet aggregation.

    PubMed

    Kuliopulos, Athan; Mohanlal, Ramon; Covic, Lidija

    2004-12-01

    Systemic inflammation has been shown to be a contributing factor to the instability of atherosclerotic plaques in patients with acute coronary syndromes (ACS). VX-702, a novel p38 mitogen-activated protein kinase (MAPK) inhibitor, is currently under investigation in ACS patients with unstable angina to evaluate its safety and efficacy during percutaneous coronary intervention (PCI). The role of p38 MAPK in platelet aggregation of normal individuals was examined using the selective second generation p38 MAPK inhibitor VX-702. Treatment of platelets with thrombin (activates PAR1 and PAR4 thrombin receptors), SFLLRN (PAR1), AYPGKF (PAR4), collagen (alpha2beta1 and GPVI/FCgammaIIR receptors) and U46619 (TXA(2)) resulted in strong activation of p38 MAPK. Activation of the GPIb von Willebrand factor receptor with ristocetin did not stimulate p38 MAPK. Pre-treatment of platelets with 1 microM VX-702 completely inhibited activation of p38 MAPK by thrombin, SFLLRN, AYPGKF, U46619, and collagen. There was no effect of VX-702 on platelet aggregation induced by any of the agonists in the presence or absence of aspirin, heparin or apyrase. It has been postulated that a potential role of p38 MAPK is to activate phospholipase A(2) (cPLA(2)) which catalyses formation of arachidonic acid leading to production of thromboxane. Interestingly, we show contrasting effects of p38 MAPK inhibition as compared to aspirin inhibition on platelet aggregation in response to collagen. Blockade of TXA(2) production by aspirin results in significant inhibition of collagen activation. However,VX-702 has no effect on collagen-mediated platelet aggregation, suggesting that blocking p38 MAPK does not effect thromboxane production in human platelets. Therefore, unlike aspirin blockade of thromboxane production in platelets, p38 MAPK inhibitors such as VX-702 do not significantly affect platelet function and would not be expected to contribute to an elevated risk of bleeding side-effects in treated

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

    SciTech Connect

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

    1989-12-15

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

  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. PMID:25994029

  16. 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.

  17. Individual dosing of ASA prophylaxis by controlling platelet aggregation.

    PubMed

    Syrbe, G; Redlich, H; Weidlich, B; Ludwig, J; Kopitzsch, S; Göckefitz, A; Herzog, T

    2001-07-01

    Acetylsalicylic acid is widely used in the primary and secondary prevention of cardiovascular diseases. In the current study, we used platelet aggregation ex vivo in platelet-rich plasma induced with arachidonic acid as a routine method for the determination of the individual dose of acetylsalicylic acid necessary to inhibit platelet aggregation in 108 patients with cardiovascular diseases. In 40% of all patients studied, a dose of 30 mg/day was sufficient to block the arachidonic acid-induced platelet aggregation nearly completely. In 50% of all patients, a dose of 100 mg/day was necessary. In 10% of all patients, the dose had to be further increased to 300 mg/day or even to 500 mg/day to inhibit platelet aggregation nearly completely. These results demonstrate that platelet aggregation can be used as a simple routine laboratory method to control acetylsalicylic acid treatment in patients with cardiovascular diseases and to determine individual doses of acetylsalicylic acid for a nearly complete inhibition of platelet aggregation. With a standard dose of 100 mg/day, 10% of the patients were nonresponders. PMID:11441981

  18. 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

  19. 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

  20. 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. PMID:16619332

  1. 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

  2. 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

  3. Ajoene inhibition of platelet aggregation: possible mediation by a hemoprotein.

    PubMed

    Jamaluddin, M P; Krishnan, L K; Thomas, A

    1988-05-31

    Ajoene, an organosulfur compound derived from garlic, was found by spectral measurements, to interact, cooperatively, with a purified hemoprotein implicated, previously, in platelet activation. It modified the binding interactions of the protein with ligands, deemed to be physiologically relevant as effectors. The characteristics of the modifications were found to parallel those of ajoene induced modifications of agonist-induced aggregation kinetics of gel-filtered calf platelets.

  4. 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. PMID:26515635

  5. Platelet leukocyte aggregates and markers of platelet aggregation, immune activation and disease progression in HIV infected treatment naive asymptomatic individuals.

    PubMed

    Nkambule, Bongani B; Davison, Glenda; Ipp, Hayley

    2015-11-01

    Platelet aggregates play a crucial role in the immune defence mechanism against viruses. Increased levels of lipopolysaccharide have been reported in human immunodeficiency virus (HIV) infected individuals. Platelets are capable of interacting with bacterial LPS and subsequently forming platelet leukocyte aggregates (PLAs). This study aimed at determining the levels of circulating PLAs in treatment naïve HIV infected individuals and correlating them, with markers of immune activation, disease progression and platelet aggregation. Thirty-two HIV negative and 35 HIV positive individuals were recruited from a clinic in the Western Cape. Platelet monocyte and platelet neutrophil aggregates were measured using flow cytometry at baseline and were correlated with markers of platelet activation (CD62P); aggregation (CD36); monocyte and neutrophil activation (CD69); monocyte tissue factor expression (CD142); immune activation (CD38 on T+ cells); D-dimers (a marker of active coagulation); CD4 count and viral load. Platelet monocyte aggregates were also measured post stimulation with lipopolysaccharide. PMA levels were higher in HIV 25.26 (16.16-32.28) versus control 14.12 (8.36-18.83), p = 0.0001. PMAs correlated with %CD38/8 expression (r = 0.54624, p = 0.0155); CD4 count (r = -0.6964, p = 0.0039) viral load (r = 0.633, p < 0.009) and monocyte %CD69 expression (r = 0.757, p = 0.030). In addition the %PMAs correlated with platelet %CD36 (r = 0.606, p = 0.017). The HIV group showed increased levels of %CD62P 5.44 (2.72-11.87) versus control 1.15 (0.19-3.59), p < 0.0001; %CD36 22.53 (10.59-55.15) versus 11.01 (3.69-26.98), p = 0.0312 and tissue factor (CD142) MFI 4.84 (4.01-8.17) versus 1.74 (1.07-9.3), p = 0.0240. We describe increased levels of circulating PMAs which directly correlates with markers of immune activation, disease progression and platelet aggregation in HIV treatment naïve individuals.

  6. Platelet-activating factor: mediator of the third pathway of platelet aggregation? A study in three patients with deficient platelet-activating factor synthesis.

    PubMed Central

    Sturk, A; Schaap, M C; ten Cate, J W; Heymans, H S; Schutgens, R B; Przyrembel, H; Borst, P

    1987-01-01

    Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway. PMID:3805272

  7. Inhibitors of platelet lipoxygenase from Ponkan fruit.

    PubMed

    Nogata, Y; Sekiya, K; Ohta, H; Kusumoto, K; Ishizu, T

    2001-04-01

    An activity-guided separation for inhibitors of rat platelet 12-lipoxygenase led to the isolation of two compounds, 4-O-feruloyl-5-O-caffeoylquinic acid (IC50; 5.5 microM) and methyl 4-O-feruloyl-5-O-caffeoylquinate (IC50; 1.9 microM) from the peel of Ponkan fruit (Citrus reticulata). The complete structure of each phenolic ester was determined by NMR spectroscopy [1H and 13C NMR spectra, 1H-1H correlation spectroscopy (COSY), 1H-detected heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond connectivity (HMBC) spectroscopies] and other spectral methods. PMID:11314960

  8. Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure.

    PubMed

    Apitz-Castro, R; Cabrera, S; Cruz, M R; Ledezma, E; Jain, M K

    1983-10-15

    We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide. PMID:6419374

  9. Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation.

    PubMed

    Shen, Ming-Yi; Chen, Fang-Yu; Hsu, Jing-Fang; Fu, Ru-Huei; Chang, Chia-Ming; Chang, Chiz-Tzung; Liu, Chung-Hsiang; Wu, Jia-Rong; Lee, An-Sheng; Chan, Hua-Chen; Sheu, Joen-Rong; Lin, Shinn-Zong; Shyu, Woei-Cherng; Sawamura, Tatsuya; Chang, Kuan-Cheng; Hsu, Chung Y; Chen, Chu-Huang

    2016-03-10

    L5, the most electronegative and atherogenic subfraction of low-density lipoprotein (LDL), induces platelet activation. We hypothesized that plasma L5 levels are increased in acute ischemic stroke patients and examined whether lectin-like oxidized LDL receptor-1 (LOX-1), the receptor for L5 on endothelial cells and platelets, plays a critical role in stroke. Because amyloid β (Aβ) stimulates platelet aggregation, we studied whether L5 and Aβ function synergistically to induce prothrombotic pathways leading to stroke. Levels of plasma L5, serum Aβ, and platelet LOX-1 expression were significantly higher in acute ischemic stroke patients than in controls without metabolic syndrome (P < .01). In mice subjected to focal cerebral ischemia, L5 treatment resulted in larger infarction volumes than did phosphate-buffered saline treatment. Deficiency or neutralizing of LOX-1 reduced infarct volume up to threefold after focal cerebral ischemia in mice, illustrating the importance of LOX-1 in stroke injury. In human platelets, L5 but not L1 (the least electronegative LDL subfraction) induced Aβ release via IκB kinase 2 (IKK2). Furthermore, L5+Aβ synergistically induced glycoprotein IIb/IIIa receptor activation; phosphorylation of IKK2, IκBα, p65, and c-Jun N-terminal kinase 1; and platelet aggregation. These effects were blocked by inhibiting IKK2, LOX-1, or nuclear factor-κB (NF-κB). Injecting L5+Aβ shortened tail-bleeding time by 50% (n = 12; P < .05 vs L1-injected mice), which was prevented by the IKK2 inhibitor. Our findings suggest that, through LOX-1, atherogenic L5 potentiates Aβ-mediated platelet activation, platelet aggregation, and hemostasis via IKK2/NF-κB signaling. L5 elevation may be a risk factor for cerebral atherothrombosis, and downregulating LOX-1 and inhibiting IKK2 may be novel antithrombotic strategies. PMID:26679863

  10. Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation

    PubMed Central

    Shen, Ming-Yi; Chen, Fang-Yu; Hsu, Jing-Fang; Fu, Ru-Huei; Chang, Chia-Ming; Chang, Chiz-Tzung; Liu, Chung-Hsiang; Wu, Jia-Rong; Lee, An-Sheng; Chan, Hua-Chen; Sheu, Joen-Rong; Lin, Shinn-Zong; Shyu, Woei-Cherng; Sawamura, Tatsuya; Chang, Kuan-Cheng; Hsu, Chung Y.

    2016-01-01

    L5, the most electronegative and atherogenic subfraction of low-density lipoprotein (LDL), induces platelet activation. We hypothesized that plasma L5 levels are increased in acute ischemic stroke patients and examined whether lectin-like oxidized LDL receptor-1 (LOX-1), the receptor for L5 on endothelial cells and platelets, plays a critical role in stroke. Because amyloid β (Aβ) stimulates platelet aggregation, we studied whether L5 and Aβ function synergistically to induce prothrombotic pathways leading to stroke. Levels of plasma L5, serum Aβ, and platelet LOX-1 expression were significantly higher in acute ischemic stroke patients than in controls without metabolic syndrome (P < .01). In mice subjected to focal cerebral ischemia, L5 treatment resulted in larger infarction volumes than did phosphate-buffered saline treatment. Deficiency or neutralizing of LOX-1 reduced infarct volume up to threefold after focal cerebral ischemia in mice, illustrating the importance of LOX-1 in stroke injury. In human platelets, L5 but not L1 (the least electronegative LDL subfraction) induced Aβ release via IκB kinase 2 (IKK2). Furthermore, L5+Aβ synergistically induced glycoprotein IIb/IIIa receptor activation; phosphorylation of IKK2, IκBα, p65, and c-Jun N-terminal kinase 1; and platelet aggregation. These effects were blocked by inhibiting IKK2, LOX-1, or nuclear factor–κB (NF-κB). Injecting L5+Aβ shortened tail-bleeding time by 50% (n = 12; P < .05 vs L1-injected mice), which was prevented by the IKK2 inhibitor. Our findings suggest that, through LOX-1, atherogenic L5 potentiates Aβ-mediated platelet activation, platelet aggregation, and hemostasis via IKK2/NF-κB signaling. L5 elevation may be a risk factor for cerebral atherothrombosis, and downregulating LOX-1 and inhibiting IKK2 may be novel antithrombotic strategies. PMID:26679863

  11. Platelet activation and platelet-monocyte aggregate formation contribute to decreased platelet count during acute simian immunodeficiency virus infection in pig-tailed macaques.

    PubMed

    Metcalf Pate, Kelly A; Lyons, Claire E; Dorsey, Jamie L; Shirk, Erin N; Queen, Suzanne E; Adams, Robert J; Gama, Lucio; Morrell, Craig N; Mankowski, Joseph L

    2013-09-01

    Platelets are key participants in innate immune responses to pathogens. As a decrease in circulating platelet count is one of the initial hematologic indicators of human immunodeficiency virus (HIV) infection, we sought to determine whether decline in platelet number during acute infection results from decreased production, increased antibody-mediated destruction, or increased platelet activation in a simian immunodeficiency virus (SIV)/macaque model. During acute SIV infection, circulating platelets were activated with increased surface expression of P-selection, CD40L and major histocompatibility complex class I. Platelet production was maintained and platelet autoantibodies were not detected during acute infection. Concurrent with a decrease in platelet numbers and an increase in circulating monocytes, platelets were found sequestered in platelet-monocyte aggregates, thereby contributing to the decline in platelet counts. Because the majority of circulating CD16(+) monocytes formed complexes with platelets during acute SIV infection, a decreased platelet count may represent platelet participation in the innate immune response to HIV.

  12. Differential inhibition of tumour cell-induced platelet aggregation by the nicotinate aspirin prodrug (ST0702) and aspirin

    PubMed Central

    Medina, Carlos; Harmon, Shona; Inkielewicz, Iwona; Santos-Martinez, Maria Jose; Jones, Michael; Cantwell, Paula; Bazou, Despina; Ledwidge, Mark; Radomski, Marek W; Gilmer, John F

    2012-01-01

    BACKGROUND AND PURPOSE Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA. EXPERIMENTAL APPROACH TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively. KEY RESULTS Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA. CONCLUSIONS AND IMPLICATIONS Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA. PMID:22122360

  13. 21 CFR 864.5700 - Automated platelet aggregation system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 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, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 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. 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...

  16. 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...

  17. 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...

  18. Antithrombotic activity of Vitis labrusca extract on rat platelet aggregation.

    PubMed

    Kwon, Se-Uk; Lee, Hoon-Yeon; Xin, Mingjie; Ji, Su-Jeong; Cho, Hyoung-Kwon; Kim, Dae-Sung; Kim, Dae-Ki; Lee, Young-Mi

    2016-03-01

    Vitis labrusca is a grapevine that has antioxidant, neuroprotective, hepatoprotective, and anticarcinogenic activity. However, the antithrombotic effect of Vitis labrusca leaves on platelets is yet to be ascertained. We investigated the inhibitory effect of V. labrusca leaf extract (VLE) on platelet aggregation in vitro and ex vivo. The thromboxane B2 (TXB2) and serotonin concentrations were measured by ELISA. The flavonoids content was measured by ultraperformance liquid chromatography (UPLC). The antithrombotic activity of VLE was evaluated using various agonists in vitro. VLE strongly inhibited adenosine diphosphate (ADP)-induced platelet aggregation. In rats, VLE treatment (100 mg/kg) reduced ADP-stimulated platelet aggregation, without affecting tail bleeding and coagulation time. Moreover, VLE significantly suppressed TXB2 and serotonin secretion. UPLC analysis indicated that VLE contains quercetin, isorhamnetin, and rutin. Our results indicate that VLE possesses antiplatelet activity via the suppression of TXB2 and serotonin, without affecting bleeding. Further, we identified the flavonoids present in VLE. Thus, VLE may be a potential agent for the prevention of cardiovascular diseases. PMID:26340455

  19. 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. PMID:23070864

  20. 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.

  1. Aspirin sensitivity of platelet aggregation in diabetes mellitus.

    PubMed

    Albert, Stewart G; Hasnain, Bibi I; Ritter, Detlef G; Joist, J Heinrich; Mooradian, Arshag D

    2005-11-01

    Although aspirin is cardioprotective in high-risk populations, many with diabetes mellitus (DM) are unresponsive to these benefits. We questioned whether cardiovascular unresponsiveness might be demonstrated by lack of aspirin sensitivity to in vitro platelet functions especially in subjects with poorly controlled diabetes. Six women and 4 men (48+/-8 years [mean+/-S.D.]), selected for poor control (glycohemoglobin 11.9+/-2.2%) and 10 sex-age (+/-5 years) matched controls received 81 mg aspirin daily. There was a 2-week washout from aspirin and related drugs. After the aspirin dose on day-7, blood for platelet aggregation assays, and 24-h urine for 2,3 dinor thromboxane B2 (TxB2) and 2,3 dinor 6-keto (PGF1alpha) were obtained. Aspirin sensitivity was defined as inhibition (i.e., lower than expected) platelet aggregation after exposure to an agonist. Those with diabetes and controls were sensitive to aspirin inhibition of platelet aggregation induced by 1.6 mM arachidonic acid (9.5+/-3.9% versus 9.1+/-3.1%, normal range 40-100%) and by 0.83 microg/mL collagen (17.4+/-13.9% versus 13.2+/-9.3%, normal range 60-93%), respectively. Aspirin sensitivity to 2 microM ADP was present in five with diabetes and five controls. Urinary prostaglandin metabolites were suppressed below reference ranges, without differences between those with DM or controls for TxB2 (350+/-149 pg/mg versus 348+/-93 pg/mg creatinine) and PGF1alpha (255+/-104 pg/mg versus 222+/-88 pg/mg creatinine). In conclusion, in poorly controlled diabetes, there was no differential lack of aspirin sensitivity to platelet aggregation, or lack of aspirin suppression of urinary TxB2 or PGF1alpha, compared with controls on aspirin. Despite suppression of urinary prostaglandin metabolites, aspirin resistance was most apparent to ADP-mediated platelet aggregation. It is not known what level of inhibition of in vitro tests is necessary for the cardioprotective benefits of aspirin in diabetes mellitus. Thus, the lack of

  2. Scalable evaluation of platelet aggregation by the degree of blood migration

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

    Platelet aggregation plays a key role in vascular thrombosis. Antiplatelet drug therapy is commonly used for the prevention of abnormal platelet aggregation. So, measuring platelet aggregation function is critically important in clinical field. Here, we introduce a scalable evaluation method of platelet aggregation measured with the degree of blood migration through microchannel in a microfluidic chip. Unlike conventional methods that require expertise with system physics to operate devices, our approach is using microfluidics system, which requires only a syringe vacuum. The scalable migration factors, migration distance and touchdown time, are capable of distinguishing various antiplatelet drug effects under microfluidics and would be effective for the quick and easy evaluation of quantitative platelet aggregation.

  3. 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.

  4. von Willebrand factor binds to platelets and induces aggregation in platelet-type but not type IIB von Willebrand disease.

    PubMed Central

    Miller, J L; Kupinski, J M; Castella, A; Ruggeri, Z M

    1983-01-01

    Platelet-type von Willebrand disease (vWD) and pseudo-vWD are two recently described intrinsic platelet defects characterized by enhanced ristocetin-induced agglutination in platelet-rich plasma. A similar finding is also typical of type IIB vWD, where it has been related to a von Willebrand factor (vWF) rather than a platelet abnormality. Platelet aggregation induced by unmodified human vWF in the absence of other stimuli has been reported in pseudo-vWD. In this study we demonstrate that vWF induces aggregation in platelet-type but not type IIB vWD. Aggregation is observed when normal plasma cryoprecipitate or purified vWF are added to platelet-rich plasma. Cryoprecipitate also aggregates washed platelets, although at higher concentrations than required for platelet-rich plasma. Purified vWF, however, induces significant aggregation of washed platelets only when plasma is added. EDTA inhibits vWF-induced aggregation. Its effect can be overcome by calcium but much less effectively by magnesium ions. Unstimulated platelets in platelet-rich plasma from patients with platelet-type but not type IIB vWD bind 125I-vWF in a specific and saturable manner. All different sized multimers of vWF become associated with platelets. Both aggregation and binding exhibit a similar vWF concentration dependence, suggesting that a correlation exists between these two events. Removal of ADP by appropriate consuming systems is without effect upon such binding or upon vWF-induced aggregation. Thrombin-induced 125I-vWF binding to washed platelets is normal in platelet-type as well as type IIB vWD. These results demonstrate that a specific binding site for unmodified human vWF is exposed on unstimulated platelets in platelet-type vWD. The relatively high vWF concentrations required for aggregation and binding may explain the lack of significant in vivo aggregation and thrombocytopenia in these patients. Moreover, these studies provide additional evidence that platelet-type and type IIB v

  5. 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. PMID:15325737

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

    PubMed

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

    2012-01-01

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

  7. Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial.

    PubMed

    Spiel, Alexander O; Bartko, Johann; Schwameis, Michael; Firbas, Christa; Siller-Matula, Jolanta; Schuetz, Matthias; Weigl, Manuela; Jilma, Bernd

    2011-04-01

    Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events. PMID:21301783

  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. Thioredoxin Inhibitors Attenuate Platelet Function and Thrombus Formation

    PubMed Central

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

    2016-01-01

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

  10. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC.

  11. Staphylococcal enterotoxin B initiates protein kinase C translocation and eicosanoid metabolism while inhibiting thrombin-induced aggregation in human platelets.

    PubMed

    Tran, Uyen; Boyle, Thomas; Shupp, Jeffrey W; Hammamieh, Rasha; Jett, Marti

    2006-08-01

    Staphylococcal enterotoxin (SE) B, a heat-stable toxin secreted by Staphylococcus aureus, has been implicated in the pathogenesis and exacerbation of several critical illnesses. It has been hypothesized that enterotoxins may interact with blood products such as platelets, in addition to T-lymphocytes and renal proximal tubule cells. The aim of this present study was to elucidate whether SEB directly alters human platelet function. Human platelet rich plasma (PRP) was pre-incubated with SEA, SEB, SEC or TSST-1, (at various concentrations and incubation times). After incubation, PRP was exposed to thrombin and aggregation was assessed. Incubation with all toxins tested resulted in decreased aggregation, specifically; exposure to 10mu g/ml of SEB for 30 min caused a 20% decrease and a 49% decrease at 90 min. A similar reduction in aggregation was seen in samples incubated with phorbol myristate acetate, a known stimulator of protein kinase C (PKC). Further, platelets exposed to SEB exhibited an increased plasma membrane PKC activity. Sphingosine, an inhibitor of PKC proved to block the SEB-induced reduction in aggregation. SEB effects on platelet metabolism were investigated using high performance liquid chromatography showing up to a 2-fold increase of active metabolites lipoxin A4 and 12-HETE, as compared to control. These data indicate that SEB is able to induce platelet dysfunction, and these effects may be mediated through activation of PKC. PMID:16550298

  12. Investigation of cyclooxygenase and signaling pathways involved in human platelet aggregation mediated by synergistic interaction of various agonists

    PubMed Central

    Khan, Nadia; Farooq, Ahsana Dar; Sadek, Bassem

    2015-01-01

    In the present study, the mechanism(s) of synergistic interaction of various platelet mediators such as arachidonic acid (AA) when combined with 5-hydroxytryptamine (5-HT) or adenosine diphosphate (ADP) on human platelet aggregation were examined. The results demonstrated that 5-HT had no or negligible effect on aggregation but it did potentiate the aggregation response of AA. Similarly, the combination of subeffective concentrations of ADP and AA exhibited noticeable rise in platelet aggregation. Moreover, the observed synergistic effect of AA with 5-HT on platelets was inhibited by different cyclooxygenase (COX) inhibitors, namely ibuprofen and celecoxib, with half maximal inhibitory effect (IC50) values of 18.0±1.8 and 15.6±3.4 μmol/L, respectively. Interestingly, the synergistic effect observed for AA with 5-HT was, also, blocked by the 5-HT receptor blockers cyproheptadine (IC50=22.0±7 μmol/L), ketanserin (IC50=152±23 μmol/L), phospholipase C (PLC) inhibitor (U73122; IC50=6.1±0.8 μmol/L), and mitogen activated protein kinase (MAPK) inhibitor (PD98059; IC50=3.8±0.5 μmol/L). Likewise, the synergism of AA and ADP was, also, attenuated by COX inhibitors (ibuprofen; IC50=20±4 μmol/L and celecoxib; IC50=24±7 μmol/L), PLC inhibitor (U73122; IC50=3.7±0.3 μmol/L), and MAPK inhibitor (PD98059; IC50=2.8±1.1 μmol/L). Our observed data demonstrate that the combination of subthreshold concentrations of agonists amplifies platelet aggregation and that these synergistic effects largely depend on activation of COX/thromboxane A2, receptor-operated Ca2+ channels, Gq/PLC, and MAPK signaling pathways. Moreover, our data revealed that inhibition of COX pathways by using both selective and/or non-selective COX inhibitors blocks not only AA metabolism and thromboxane A2 formation, but also its binding to Gq receptors and activation of receptor-operated Ca2+ channels in platelets. Overall, our results show that PLC and MAPK inhibitors proved to inhibit the

  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. Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

    PubMed

    Dangelmaier, Carol A; Holmsen, Holm

    2014-01-01

    Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

  15. Effect of supine exercise on platelet aggregation and fibrinolytic activity.

    PubMed

    Dag, B; Gleerup, G; Bak, A M; Hindberg, I; Mehlsen, J; Winther, K

    1994-03-01

    In 12 healthy young men, strenuous cycling exercise in the supine position, caused platelet aggregability to decrease and the ADP threshold to rise from 7.0 microM resting, to 9.5 exercising (P < 0.01). At the same time, fibrinolytic activity increased markedly: euglobulin clot lysis time shortened from 178 to 68 min, PAI-1 fell from 8.91 to 5.16 IU ml-1, and t-PA rose from 0.56 to 3.95 IU ml-1, all three values were significant to P < 0.01. When the erect posture was assumed after lying at ease for 1 h after exercise, it did not increase platelet activity as expected, but caused a modest increase of fibrinolytic activity. These results suggest that supine exercise will not affect the haemostatic system adversely.

  16. Amorphous silica nanoparticles aggregate human platelets: potential implications for vascular homeostasis

    PubMed Central

    Corbalan, J Jose; Medina, Carlos; Jacoby, Adam; Malinski, Tadeusz; Radomski, Marek W

    2012-01-01

    Background Amorphous silica nanoparticles (SiNP) can be used in medical technologies and other industries leading to human exposure. However, an increased number of studies indicate that this exposure may result in cardiovascular inflammation and damage. A high ratio of nitric oxide to peroxynitrite concentrations ([NO]/[ONOO−]) is crucial for cardiovascular homeostasis and platelet hemostasis. Therefore, we studied the influence of SiNP on the platelet [NO]/[ONOO−] balance and platelet aggregation. Methods Nanoparticle–platelet interaction was examined using transmission electron microscopy. Electrochemical nanosensors were used to measure the levels of NO and ONOO− released by platelets upon nanoparticle stimulation. Platelet aggregation was studied using light aggregometry, flow cytometry, and phase contrast microscopy. Results Amorphous SiNP induced NO release from platelets followed by a massive stimulation of ONOO− leading to an unfavorably low [NO]/[ONOO−] ratio. In addition, SiNP induced an upregulation of selectin P expression and glycoprotein IIb/IIIa activation on the platelet surface membrane, and led to platelet aggregation via adenosine diphosphate and matrix metalloproteinase 2-dependent mechanisms. Importantly, all the effects on platelet aggregation were inversely proportional to nanoparticle size. Conclusions The exposure of platelets to amorphous SiNP induces a critically low [NO]/[ONOO−] ratio leading to platelet aggregation. These findings provide new insights into the pharmacological profile of SiNP in platelets. PMID:22334785

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

    PubMed

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

    2010-02-10

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

  18. Multiple electrode aggregometry: a new device to measure platelet aggregation in whole blood.

    PubMed

    Tóth, Orsolya; Calatzis, Andreas; Penz, Sandra; Losonczy, Hajna; Siess, Wolfgang

    2006-12-01

    Several methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p < 0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications. PMID:17139373

  19. Structural determinants of Tau aggregation inhibitor potency.

    PubMed

    Schafer, Kelsey N; Cisek, Katryna; Huseby, Carol J; Chang, Edward; Kuret, Jeff

    2013-11-01

    Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers.

  20. Structural Determinants of Tau Aggregation Inhibitor Potency*

    PubMed Central

    Schafer, Kelsey N.; Cisek, Katryna; Huseby, Carol J.; Chang, Edward; Kuret, Jeff

    2013-01-01

    Small-molecule Tau aggregation inhibitors are under investigation as potential therapeutic agents against Alzheimer disease. Many such inhibitors have been identified in vitro, but their potency-driving features, and their molecular targets in the Tau aggregation pathway, have resisted identification. Previously we proposed ligand polarizability, a measure of electron delocalization, as a candidate descriptor of inhibitor potency. Here we tested this hypothesis by correlating the ground state polarizabilities of cyanine, phenothiazine, and arylmethine derivatives calculated using ab initio quantum methods with inhibitory potency values determined in the presence of octadecyl sulfate inducer under reducing conditions. A series of rhodanine analogs was analyzed as well using potency values disclosed in the literature. Results showed that polarizability and inhibitory potency directly correlated within all four series. To identify putative binding targets, representative members of the four chemotypes were added to aggregation reactions, where they were found to stabilize soluble, but SDS-resistant Tau species at the expense of filamentous aggregates. Using SDS resistance as a secondary assay, and a library of Tau deletion and missense mutants as targets, interaction with cyanine was localized to the microtubule binding repeat region. Moreover, the SDS-resistant phenotype was completely dependent on the presence of octadecyl sulfate inducer, but not intact PHF6/PH6* hexapeptide motifs, indicating that cyanine interacted with a species in the aggregation pathway prior to nucleus formation. Together the data suggest that flat, highly polarizable ligands inhibit Tau aggregation by interacting with folded species in the aggregation pathway and driving their assembly into soluble but highly stable Tau oligomers. PMID:24072703

  1. [Effect of troxerutin and cerebroproptein hydrolysate injection on platelet aggregation and thrombosis].

    PubMed

    Chen, Qiu-Chen; Yu, Zhao-Jin; Sun, Hai-Gang; Yu, Jian-Kun; Wei, Min-Jie

    2011-02-01

    This study was purposed to explore the effect of troxerotin and cerebroproptein hydrolysate injection (TCHI) on platelet aggregation in vitro and thrombosis in vivo. The inhibitory rate of TCHI at different concentrations on platelet aggregation was determined by platelet aggregometer. The relationship between dose and effect was established. The effect of troxerutin and cerebroproptein hydrolysate injection on thrombosis was determined by the carotid thrombosis model of rats. The results showed that the TCHI could inhibit thrombosis and platelet aggregation in a concentration-dependent way. When the concentration of TCHI total nitrogen was 5 µg/ml, the inhibition rate of platelet aggregation reached to the highest value of 28.61 ± 22.07%, which is 2.5 times as much as that with 100 µg/ml aspirin. It is concluded that the TCHI has antiaggregative and antithrombotic activity effects against platelet aggregation and thrombosis.

  2. Involvement of Nitric Oxide on Calcium Mobilization and Arachidonic Acid Pathway Activation during Platelet Aggregation with different aggregating agonists

    PubMed Central

    Banerjee, Debipriya; Mazumder, Sahana; Kumar Sinha, Asru

    2016-01-01

    Platelet aggregation by different aggregating agonists is essential in the normal blood coagulation process, the excess of which caused acute coronary syndrome (ACS). In all cases, the activation of arachidonic acid by cycloxygenase was needed for the synthesis of thromboxane A2 (TXA2) but the mechanism of arachidonic acid release in platelets remains obscure. Studies were conducted to determine the role of nitric oxide (NO), if any, on the release of arachidonic acid in platelets. The cytosolic Ca2+ was visualized and quantitated by fluorescent spectroscopy by using QUIN-2. NO was measured by methemoglobin method. Arachidonic acid was determined by HPLC. TXA2 was measured as ThromboxaneB2 (TXB2) by ELISA. Treatment of platelets in platelet-rich plasma (PRP) with different aggregating agents resulted in the inhibition of nitric oxide synthase (NOS) which inhibited the production of NO synthesis and increased TXA2 synthesis. Furthermore, the treatment of washed PRP with different platelet aggregating agents resulted in the increase of [Ca2+] in nM ranges. In contrast, the pre-treatment of washed PRP with aspirin increased platelet NO level and inhibited the Ca2+ mobilization and TXA2 synthesis. These results indicated that the aggregation of platelets by different aggregating agonists was caused by the cytosolic Ca2+ mobilization due to the inhibition of NOS. PMID:27127451

  3. [Platelet aggregation upon acetylsalicylic acid and clopidogrel treatment and glycoprotein IIb/IIIa content in patients with acute coronary syndrome].

    PubMed

    Khaspekova, S G; Ziuriaev, I T; Iakushkin, V V; Golubeva, N V; Ruda, M Ia; Mazurov, A V

    2011-01-01

    Interaction between aggregating activity of platelets and glycoprotein (GP) IIb/IIIa (fibrinogen receptor) content on their surface was investigated in patients with acute coronary syndrome (ACS). Eighty nine ACS patients were included into the study - 69 with and 20 without elevation of ST segment. Blood was collected within the first hour of admission to the clinic (1 day), and then at 3-5 and 8-12 days. All patients received standard antiaggregant therapy - acetylsalicylic acid - ASA (thromboxane A2 synthesis inhibitor) and clopidogrel (ADP receptor antagonist). Platelet aggregation was analyzed at the first time point when patients had already taken ASA but not clopidogrel, and then (3-5 and 8- 12 days) upon combined therapy with both preparations. Aggregation was induced by 5 and 20 uM ADP and measured by turbidimetric method. In comparison with the initial level (1 day, ASA) at days 3-5, i.e. after development of clopidogrel effect, platelet aggregation was decreased by 54 and 40% upon its stimulation with 5 and 20 uM ADP, and was not further changed at days 8-12. GP IIb/IIIa content on platelet surface was determined by binding of 125I-labelled monoclonal antibody CRC64. GP IIb/IIIa number varied from 31100 to 73000 per platelet with the mean level of 48500 +/- 8400 (mean +/- standard deviation). No differences were detected between mean GP IIb/IIIa number at 1, 3-5 and 8-12 days after ACS onset. Upon repeat GP IIb/IIIa measurement coefficient of variation was 6.1% demonstrating the stability of this parameter in each patient. Positive correlation between platelet aggregation and GP IIb/IIIa content was detected at the first day - correlation coefficients (r) 0.425 and 0.470 for 5 and 20 uM ADP (n=57, p<0.001). However positive association between these parameters was not revealed at 3-5 and 8-12 days, when patients received not only ASA but clopidogrel as well (r from -0.054 to -0.237, p>0.05). These results indicates that variations of GP IIb/IIIa content

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

    PubMed Central

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

    2016-01-01

    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. PMID:27432161

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

    PubMed

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

    2016-04-01

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

  6. Mediterranean wild plants reduce postprandial platelet aggregation in patients with metabolic syndrome.

    PubMed

    Fragopoulou, Elizabeth; Detopoulou, Paraskevi; Nomikos, Tzortzis; Pliakis, Emmanuel; Panagiotakos, Demosthenes B; Antonopoulou, Smaragdi

    2012-03-01

    Postprandial platelet hyperactivity and aggregation play a crucial role in the pathogenesis of metabolic syndrome. The purpose of the present study was to evaluate the effect of boiled wild plants consumption on the postprandial platelet aggregation in metabolic syndrome patients. Patients consumed 5 meals in a random order (ie, 4 wild plant meals, namely, Reichardia picroides [RP], Cynara cardunculus, Urospermum picroides [UP], and Chrysanthemum coronarium, and a control meal, which contained no wild plants). Several biochemical indices as well as platelet activating factor (PAF)- and adenosine diphosphate-induced ex vivo platelet aggregation were measured postprandially. Moreover, the ability of plants extract to inhibit rabbit platelet aggregation was tested in vitro. The consumption of RP and UP meals significantly reduced ex vivo adenosine diphosphate-induced postprandial platelet aggregation compared with the control meal. The consumption of UP meals significantly reduced the ex vivo PAF-induced platelet aggregation postprandially. Both UP and RP extracts significantly inhibited PAF-induced rabbit platelet aggregation in vitro. Wild plants consumption reduced postprandial platelet hyperaggregability of metabolic syndrome patients, which may account for their healthy effects. PMID:21944262

  7. 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

  8. 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...

  9. 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.

  10. 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.

  11. A serotonin-induced N-glycan switch regulates platelet aggregation

    PubMed Central

    Mercado, Charles P.; Quintero, Maritza V.; Li, Yicong; Singh, Preeti; Byrd, Alicia K.; Talabnin, Krajang; Ishihara, Mayumi; Azadi, Parastoo; Rusch, Nancy J.; Kuberan, Balagurunathan; Maroteaux, Luc; Kilic, Fusun

    2013-01-01

    Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associated with platelet aggregation. Mass spectrometry of total surface glycoproteins on platelets isolated from wild-type mice infused for 24 hours with saline or 5-HT revealed that the content of glycoproteins on platelets from 5-HT-infused mice switched from predominantly N-acetyl-neuraminic acid (Neu5Ac) to N-glycolyl-neuraminic acid (Neu5Gc). Cytidine monophosphate-N-acetylneuraminate hydroxylase (CMAH) synthesizes Neu5Gc from Neu5Ac. Up-regulation of Neu5Gc content on the platelet surface resulted from an increase in the catalytic function, not expression, of CMAH in platelets of 5-HT-infused mice. The highest level of Neu5Gc was observed in platelets of 5-HT-infused, 5-HT transporter-knock out mice, suggesting that the surface delineated 5-HT receptor on platelets may promote CMAH catalytic activity. These new findings link elevated levels of plasma 5-HT to altered platelet N-glycan content, a previously unrecognized abnormality that may favor platelet aggregation. PMID:24077408

  12. Acetyl eugenol, a component of oil of cloves (Syzygium aromaticum L.) inhibits aggregation and alters arachidonic acid metabolism in human blood platelets.

    PubMed

    Srivastava, K C; Malhotra, N

    1991-01-01

    In continuation of our studies with the oil of cloves--a common kitchen spice and a crude drug for home medicine--we have isolated yet another active component identified as acetyl eugenol (AE); the earlier reported active component being eugenol. The isolated material (IM) was found to be a potent platelet inhibitor; IM abolished arachidonate (AA)-induced aggregation at ca. 12 microM, a concentration needed to abolish the second phase of adrenaline-induced aggregation. Chemically synthesized acetyl eugenol showed similar effects on AA- and adrenaline-induced aggregation. A dose-dependent inhibition of collagen-induced aggregation was also observed. AE did not inhibit either calcium ionophore A23187- or thrombin-induced aggregation. Studies on aggregation and ATP release were done using whole blood (WB). AA-induced aggregation in WB was abolished at 3 micrograms/ml (14.6 microM) which persisted even after doubling the concentration of AA. ATP release was inhibited. Inhibition of aggregation appeared to be mediated by a combination of two effects: reduced formation of thromboxane and increased generation of 12-lipoxygenase product (12-HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxB2 formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids. PMID:2011614

  13. Fibrin formation by staphylothrombin facilitates Staphylococcus aureus-induced platelet aggregation.

    PubMed

    Vanassche, Thomas; Kauskot, Alexandre; Verhaegen, Jan; Peetermans, Willy E; van Ryn, Joanne; Schneewind, Olaf; Hoylaerts, Marc F; Verhamme, Peter

    2012-06-01

    Interactions of Staphylococcus aureus (S. aureus) and platelets play an important role in the pathogenesis of intravascular infections such as infective endocarditis (IE). A typical feature of S. aureus is the ability to generate thrombin activity through the secretion of two prothrombin activating molecules, staphylocoagulase and von Willebrand factor-binding protein (vWbp), which bind to human prothrombin to form the enzymatically active staphylothrombin complex. The role of staphylothrombin in the interaction between S. aureus and platelets has not yet been studied. We found that in contrast with thrombin, staphylothrombin did not directly activate human platelets. However, the staphylothrombin-mediated conversion of fibrinogen to fibrin initiated platelet aggregation and secondary activation and facilitated S. aureus-platelet interactions. Both the genetic absence of staphylocoagulase and vWbp and pharmacological inhibition of staphylothrombin increased the lag time to aggregation, and reduced platelet trapping by S. aureus in high shear stress conditions. The combined inhibition of staphylothrombin and immunoglobulin binding to platelets completely abolished the ability of S. aureus to aggregate platelets in vitro. In conclusion, although staphylothrombin did not directly activate platelets, the formation of a fibrin scaffold facilitated bacteria-platelet interaction, and the inhibition of staphylothrombin resulted in a reduced activation of platelets by S. aureus. PMID:22437005

  14. Lack of association between serum paraoxonase-1 activity and residual platelet aggregation during dual anti-platelet therapy.

    PubMed

    Ohmori, Tsukasa; Yano, Yuichiro; Sakata, Asuka; Ikemoto, Tomokazu; Shimpo, Masahisa; Madoiwa, Seiji; Katsuki, Takaaki; Mimuro, Jun; Shimada, Kazuyuki; Kario, Kazuomi; Sakata, Yoichi

    2012-04-01

    High residual platelet aggregability during thienopyridine treatment occurs because of low levels of the active drug metabolite, and is associated with an increased rate of major adverse cardiovascular events. Recent findings suggest that paraoxonase-1 (PON1) is a major determinant for clopidogrel efficacy. The aim of this study was to assess the impact of serum PON1 activity on platelet aggregability in thienopyridine-treated patients. In 72 patients receiving treatment with aspirin and ticlopidine after acute coronary syndrome, various laboratory data including the formation of platelet aggregations induced by agonists were compared with serum PON1 activities, measured as paraoxonase and homocysteine thiolactone hydrolase (HTLase). Serum paraoxonase activity was significantly associated with HTLase activity (R=0.4487, P<0.0001). These PON1 activities were not correlated with any parameters for platelet aggregation, hypertension, sleep apnea, and diabetes mellitus. In contrast, serum PON1 activities seemed to be involved in cardiac function, with brain natriuretic peptide and ejection fraction being significantly correlated with serum HTLase activity (R=-0.2767, P=0.0214) and paraoxonase activity (R=0.2558, P=0.0339), respectively. Paraoxonase activity also demonstrated a significant association with increased levels of ankle-brachial index (R=0.267, P=0.0255). Serum PON1 activities did not influence platelet aggregability during treatment with thienopyridine. However, they might modulate cardiac function after acute coronary syndrome and progression of atherosclerosis. PMID:22115701

  15. Platelet protective efficacy of 3,4,5 trisubstituted isoxazole analogue by inhibiting ROS-mediated apoptosis and platelet aggregation.

    PubMed

    Jagadish, Swamy; Rajeev, Narasimhamurthy; NaveenKumar, Somanathapura K; Sharath Kumar, Kothanahally S; Paul, Manoj; Hegde, Mahesh; Basappa; Sadashiva, Marilinganadoddi P; Girish, Kesturu S; Rangappa, Kanchugarakoppal S

    2016-03-01

    Thrombocytopenia is a major hematological concern in oxidative stress-associated pathologies and chronic clinical disorders, where premature platelet destruction severely affects the normal functioning of thrombosis and hemostasis. In addition, frequent exposure of platelets to chemical entities and therapeutic drugs immensely contributes in the development of thrombocytopenia leading to huge platelet loss, which might be fatal sometimes. Till date, there are only few platelet protective molecules known to combat thrombocytopenia. Hence, small molecule therapeutics are extremely in need to relieve the burden on limited treatment strategies of thrombocytopenia. In this study, we have synthesized a series of novel 3,4,5 trisubstituted isoxazole derivatives, among which compound 4a [4-methoxy-N'-(5-methyl-3-phenylisoxazole-4-carbonyl) benzenesulfonohydrazide] was found to significantly ameliorate the oxidative stress-induced platelet apoptosis by restoring various apoptotic markers such as ROS content, cytosolic Ca(2+) levels, eIF2-α phosphorylation, mitochondrial membrane depolarization, cytochrome c release, caspase activation, PS externalization, and cytotoxicity markers. Additionally, compound 4a dose dependently inhibits collagen-induced platelet aggregation. Hence, compound 4a can be considered as a prospective molecule in the treatment regime of platelet activation and apoptosis and other clinical conditions of thrombocytopenia. Further studies might ensure the use of compound 4a as a supplementary therapeutic agent to treat, thrombosis and CVD-associated complications. Over all, the study reveals a platelet protective efficacy of novel isoxazole derivative 4a with a potential to combat oxidative stress-induced platelet apoptosis.

  16. 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

  17. The effects of vincristine on platelet aggregation studied by a filter loop technique in the rat.

    PubMed Central

    Bee, D.; Leach, E.; Martin, J. F.; Suggett, A. J.

    1980-01-01

    1 A method for measuring aggregation of platelets of adenosine diphosphate (ADP) is described using a filter inserted into the flowing aortic blood in the rat. 2 Repeated infusions of ADP resulted in a fall in the calculated aggregation index without significant changes in the platelet count. 3 Vincristine (0.05 mg/kg) intravenously caused significant inhibition of ADP-induced platelet aggregation. 4 Infusion of ADP caused some peripheral vasodilatation though it is unlikely that this contributed to the effects seen to any great extent. PMID:7437636

  18. Effects of tetrandrine and fangchinoline on human platelet aggregation and thromboxane B2 formation.

    PubMed

    Kim, H S; Zhang, Y H; Fang, L H; Yun, Y P; Lee, H K

    1999-08-01

    Tetrandrine (TET) and fangchinoline (FAN) are two major components of the radix of Stephania tetrandra. The effects of TET and FAN on human platelet aggregation and formation of thromboxane (TX) B2, a stable metabolite of TXA2, were examined in the aspect of platelet aggregation. TET and FAN inhibited platelet-activating factor (PAF)-induced human platelet aggregation. IC50 values for TET and FAN were 28.6+/-3.24 microM and 21.7+/-2.61 microM, respectively. In the PAF-receptor binding assay, neither TET nor FAN showed any inhibitory effects on the specific bindings of PAF to its receptor. TET and FAN also inhibited PAF-, thrombin- and arachidonic acid-induced thromboxane B2 formation in human washed platelet. These results indicate that TET and FAN inhibit the platelet aggregation by interfering with the intracellular messengers system, but not by inhibiting the binding of PAF to PAF-receptor on the platelet membrane directly, and the suppression of TXA2 formation by TET and FAN may be responsible for their inhibitory activities on the platelet aggregation and further on the thrombosis. PMID:10433485

  19. The role of platelet aggregation and release in fragment D-induced pulmonary dysfunction.

    PubMed Central

    Manwaring, D; Curreri, P W

    1980-01-01

    The plasma concentration of fibrinogen degradation product D (fragmentt D) is markedly incrased following major burn or traumatic injury. Purified human fragment D infused into awake, restrained, nontraumatized rabbits (100 micrograms/ml blood) causes progressive thrombocytopenia, pulmonary dysfunction, vascular leak, and interstitial neutrophilia. Rabbits treated with the antihistamine diphenhydramine (Benadryl) prior to fragment D infusion fail to develop these symptoms. This study examined platelet aggregation, platelet ATP secretion, and platelet malondialdehyde release in rabbits which received fragmen D alone or fragment D following diphenhydramine pretreatment. Platelet-rich plasma was prepared from citrated blood drawn from femoral arterial catheters at 0, 2 1/2, and 4 hours postinfusion. Platelet aggregation was stimulated with either collagen or ADP. Malondialdehyde, a byproduct of thromboxane synthesis, was measured by colorimetry. Platelet aggregation and function (stimulated with collagen) were enhanced in fragment D platelet-rich plasma, since all response times decreased. Total ATP and MDA release incresed. Diphenhydramine pretreatment inhibited fragment D-enhanced aggregation, ATP release and prostaglandin (thromboxane) synthesis. No animal pretreated with diphenhydramine exhibited thrombocytopenia or respiratory dysfunction. Stimulation of platelet aggregation and release may represent one mechanism by which fragment D induces pulmonary dysfunction. Diphenhydramine inhibits these responses and may prove therapeutic in posttraumtic pulmonary complications. PMID:7406554

  20. In vitro platelet aggregation and oxidative stress caused by amorphous silica nanoparticles

    PubMed Central

    Nemmar, Abderrahim; Yuvaraju, Priya; Beegam, Sumaya; Yasin, Javed; Dhaheri, Rauda Al; Fahim, Mohamed A; Ali, Badreldin H

    2015-01-01

    Amorphous silica nanoparticles (SiNP) are being investigated for their potential use in various industrial and medical fields. Therefore, the assessment of their possible pathophysiological effect on circulating cells such as platelets is essential. We recently showed that intraperitoneal administration of SiNP causes proinflammatory and prothrombotic responses in vivo. However, little is known about the interaction of amorphous SiNP with platelets in vitro. Presently, we investigated the in vitro effects of SiNP (1, 5 and 25 μg/ml) on platelet aggregation, oxidative stress and intracellular calcium in mouse platelets. Incubation of platelets with SiNP caused a significant and dose-dependent platelet aggregation. Similarly, the activity of lactate dehydrogenase (as a marker of cell membrane integrity) was significantly increased by SiNP. Total antioxidant activity and lipid platelets vulnerability to in vitro peroxidation (measured by malondialdehyde production) were significantly increased after SiNP exposure. Additionally, SiNP exposure significantly increased the cytosolic calcium concentration. In conclusion, our in vitro data show that incubation of platelets with SiNP caused platelet aggregation, oxidative stress and increased intracellular calcium. This finding provides evidence on the toxicity of SiNP on platelet physiology. PMID:26069526

  1. 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.

  2. Silibinin: a novel inhibitor of Aβ aggregation.

    PubMed

    Yin, Fei; Liu, Jianhui; Ji, Xiuhong; Wang, Yanwen; Zidichouski, Jeffrey; Zhang, Junzeng

    2011-02-01

    Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β peptide (Aβ) into extracellular fibrillar deposits known as amyloid plaque. Inhibition of Aβ aggregation is therefore viewed as a potential method to halt or slow the progression of AD. It is reported that silibinin (silybin), a flavonoid derived from the herb milk thistle (Silybum marianum), attenuates cognitive deficits induced by Aβ25-35 peptide and methamphetamine. However, it remains unclear whether silibinin interacts with Aβ peptide directly and decreases Aβ peptide-induced neurotoxicity. In the present study, we identified, through employing a ThT assay and electron microscopic imaging that silibinin also appears to act as a novel inhibitor of Aβ aggregation and this effect showed dose-dependency. We also show that silibinin prevented SH-SY5Y cells from injuries caused by Aβ(1-42)-induced oxidative stress by decreasing H(2)O(2) production in Aβ(1-42)-stressed neurons. Taken together, these results indicate that silibinin may be a novel therapeutic agent for the treatment of AD.

  3. The effect of the menstrual cycle and of decompression stress on arachidonic acid-induced platelet aggregation and on intrinsic platelet thromboxane production in women compared with men.

    PubMed

    Markham, S M; Dubin, N H; Rock, J A

    1991-12-01

    Menstrual cycle variations in platelet aggregation and thromboxane production in association with sex steroids have been reported. External stimuli such as decompression sickness have been associated with clotting activity changes, specifically, increased platelet aggregation. Differences in response of platelets from women and men, when subjected to such a stress, have been observed. This study evaluated the ability of washed platelets from women in the proliferative and secretory phases of the menstrual cycle to aggregate in response to arachidonic acid and the aggregation difference between washed platelets from women and men in response to decompression stress and arachidonic acid. Additionally, platelet thromboxane production differences between the assessed platelet populations were compared. Our results indicate no difference in platelet aggregability between phases of the menstrual cycle. A significant aggregation difference between platelets from women and men was noted. Platelets from women were more sensitive to arachidonic acid aggregation. These differences were not affected by decompression stress. No difference in thromboxane B2 production was noted between the platelet populations evaluated.

  4. An ethanol extract of Ramulus mori improves blood circulation by inhibiting platelet aggregation.

    PubMed

    Lee, Jiyun; Kwon, Gayeung; Park, Jieun; Kim, Jeong-Keun; Choe, Soo Young; Seo, Yoonhee; Lim, Young-Hee

    2016-07-01

    Inappropriate platelet aggregation can cause blood coagulation and thrombosis. In this study, the effect of an ethanol extract of Ramulus mori (ERM) on blood circulation was investigated. The antithrombotic activity of ERM on rat carotid arterial thrombosis was evaluated in vivo, and the effect of ERM on platelet aggregation and blood coagulation time was evaluated ex vivo. To evaluate the safety of ERM, its cytotoxicity to platelets and its effect on tail bleeding time were assessed; ERM was not toxic to rat platelets and did not prolong bleeding time. Moreover, administering ERM to rats had a significant preventive effect on carotid arterial thrombosis in vivo, and significantly inhibited adenosine diphosphate- and collagen-induced platelet aggregation ex vivo, whereas it did not prolong coagulation periods, such as prothrombin time and activated partial thromboplastin time. The results suggest that ERM is effective in improving blood circulation via antiplatelet activity rather than anticoagulation activity.

  5. Platelet aggregation responses vary over a period of time in healthy controls.

    PubMed

    Refaai, Majed A; Frenkel, Eugene; Sarode, Ravi

    2010-01-01

    Platelet aggregation study is performed to investigate platelet function abnormality. A normal healthy control sample is usually run with the patient sample as a quality control measure. At our institution, we observed variations in platelet aggregation responses in our normal repeat controls. Therefore, we analysed aggregation parameters in these controls. Whole blood aggregation studies were performed with adenosine diphosphate (ADP), arachidonic acid (AA), collagen and ristocetin. Adenosine triphosphate (ATP) secretion was also measured simultaneously by leuciferin-leuciferase reaction. During a 5-year period, a total of 86 studies were performed on seven controls. Aggregations were within the acceptable range in 67% of the time. Collagen was the most affected agonist in our study. On five occasions, four controls had subnormal aggregations with two agonists. All abnormal responses were hypoaggregation except for two who had hyperaggregation with collagen and AA. Only one out of seven controls was always normal. In the presence of a subnormal control result, a new control was run before releasing the patient's platelet aggregation results. These findings suggest that many physiological factors, other than medications, may affect platelet function even in normal individuals. Therefore, a repeat study at a later date to demonstrate a reproducible abnormality would be prudent before labeling a patient's platelets abnormal.

  6. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  7. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

    PubMed

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

  8. Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

    PubMed Central

    Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

    2016-01-01

    Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

  9. On-chip evaluation of platelet adhesion and aggregation upon exposure to mesoporous silica nanoparticles.

    PubMed

    Kim, Donghyuk; Finkenstaedt-Quinn, Solaire; Hurley, Katie R; Buchman, Joseph T; Haynes, Christy L

    2014-03-01

    Mesoporous silica nanoparticles are promising drug delivery agents; however, their interaction with various in vivo biological components is still under investigation. In this work, the impact of sub-50 nm diameter mesoporous silica nanoparticles on platelet function is investigated using a microfluidic platform to model blood vessel characteristics. Platelet adhesion and aggregation in the presence of mesoporous silica nanoparticles is investigated, controlling whether or not platelets are activated ahead of nanoparticle exposure. The results indicate that nanoparticles slightly compromise platelet adhesion to endothelial cells at low nanoparticle doses, but that high nanoparticle doses significantly increase the number of platelet adhesion events, leading to higher probability for uncontrolled platelet actions (e.g. clot formation in vivo). High nanoparticle doses also induced platelet aggregation. While platelet activation and aggregation occurred, in no case did nanoparticle exposure result in significant loss of platelet viability; as such, this work clearly demonstrates that aspects besides viability, such as cellular adhesion and interaction with other cell types, have to be considered in the context of nanotoxicology. This simple and highly adaptable analytical platform will be useful for further nanotoxicity studies involving other nanoparticle and cell types.

  10. PGE(2) reverses G(s)-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-G(s)-mediated inhibition of platelet aggregation by interaction with EP4 receptors.

    PubMed

    Glenn, Jacqueline R; White, Ann E; Iyu, David; Heptinstall, Stan

    2012-01-01

    Prostaglandin E(2) (PGE(2)) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3'5'-monophosphate (cAMP). PGE(2) reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a G(s)-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE(2). Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE(2) were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through G(s)-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE(2) reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE(2) is mediated via the EP3 receptor. In contrast, PGE(2) added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE(2) is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE(2) may be prothrombotic. If so, the results described here

  11. A note on the use of Quin2 in studying shear-induced platelet aggregation.

    PubMed

    Giorgio, T D; Hellums, J D

    1986-02-01

    Quin2, a calcium ion chelator which can penetrate plasma membranes, was used to study the role of intracellular calcium ion concentration in mediating shear-induced platelet activation. Washed platelet suspensions were subjected to various levels of uniform, known shear stress in a cone and plate viscometer in the absence of added agonists. Additional samples were aggregated in response to chemical platelet agonists in a conventional aggregometer. The aggregometer response of Quin2-containing platelets to collagen, thrombin and ADP exhibited increased lag time and reduced maximum rate of aggregation in comparison to controls. However, the extent of aggregation of the Quin2-containing platelets eventually reached the same level as that of the controls. Very different results were obtained for aggregation by shear stress in the viscometer. Shear-induced aggregation was significantly suppressed by Quin2 treatment at both short (30 seconds) and long (300 seconds) times of exposure to the shear field. Shear-induced dense granular release and cellular lysis were unaltered by Quin2 treatment at 30 second exposure times, but both were significantly increased by Quin2 treatment at 300 second exposure times. These results suggest that intracellular calcium ion mobilization is an important early step in shear-induced platelet activation. Additionally, Quin2 appears to have effects resulting in increased platelet fragility. Thus, the findings raise questions on the suitability of Quin2 as an intracellular calcium ion probe in studies in shear fields. PMID:3705013

  12. gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation.

    PubMed

    Lovely, Rehana S; Rein, Chantelle M; White, Tara C; Jouihan, Sari A; Boshkov, Lynn K; Bakke, Antony C; McCarty, Owen J; Farrell, David H

    2008-11-01

    The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity. PMID:18989528

  13. Cilostazol Inhibits Accumulation of Triglyceride in Aorta and Platelet Aggregation in Cholesterol-Fed Rabbits

    PubMed Central

    Ito, Hideki; Uehara, Kenji; Matsumoto, Yutaka; Hashimoto, Ayako; Nagano, Chifumi; Niimi, Manabu; Miyakoda, Goro; Nagano, Keisuke

    2012-01-01

    Cilostazol is clinically used for the treatment of ischemic symptoms in patients with chronic peripheral arterial obstruction and for the secondary prevention of brain infarction. Recently, it has been reported that cilostazol has preventive effects on atherogenesis and decreased serum triglyceride in rodent models. There are, however, few reports on the evaluation of cilostazol using atherosclerotic rabbits, which have similar lipid metabolism to humans, and are used for investigating the lipid content in aorta and platelet aggregation under conditions of hyperlipidemia. Therefore, we evaluated the effect of cilostazol on the atherosclerosis and platelet aggregation in rabbits fed a normal diet or a cholesterol-containing diet supplemented with or without cilostazol. We evaluated the effects of cilostazol on the atherogenesis by measuring serum and aortic lipid content, and the lesion area after a 10-week treatment and the effect on platelet aggregation after 1- and 10-week treatment. From the lipid analyses, cilostazol significantly reduced the total cholesterol, triglyceride and phospholipids in serum, and moreover, the triglyceride content in the atherosclerotic aorta. Cilostazol significantly reduced the intimal atherosclerotic area. Platelet aggregation was enhanced in cholesterol-fed rabbits. Cilostazol significantly inhibited the platelet aggregation in rabbits fed both a normal diet and a high cholesterol diet. Cilostazol showed anti-atherosclerotic and anti-platelet effects in cholesterol-fed rabbits possibly due to the improvement of lipid metabolism and the attenuation of platelet activation. The results suggest that cilostazol is useful for prevention and treatment of atherothrombotic diseases with the lipid abnormalities. PMID:22761774

  14. Phase analysis of platelet aggregation in acute disturbances of cerebral circulation.

    PubMed

    Petrova, T R; Pavlishchuk, S A; Grigoriev, G I

    1975-01-01

    In 120 patients with atherosclerosis, complicated in 43 patients by a haemorrhagic, in 47 patients by an ischaemic, and in 30 patients by a transient cerebral insult, phase analysis of platelet aggregation was performed by the turbidimetric method according to Born with graphic recording according to O'Brien. An increase in the platelet activity was found in ischaemic insult, manifesting itself by the occurrence of spontaneous aggregationin 60% of the cases, an acceleration of ADP-induced aggregation, and the second aggregation phase in all patients examined. A direct correlation was revealed between the secondary aggregation and the intensity of spontaneous and of ADP-induced aggregation, and the possibility of a transformation of the spontaneous into the secondary aggregation of platelets was demonstrated. Haemorrhagic insults were characterized by the absence of spontaneous and secondary aggregation and by the suppression of ADP-induced aggregation. In a transient insult, the mean values of the aggregatogram items did differ from normal. In vitro, the role of increased permeability of platelet membranes in the mechanism triggering off spontaneous aggregation and the second phase of ADP-induced aggregation was documented.

  15. Platelet aggregation in Finnish men and its relation to fatty acids in platelets, plasma and adipose tissue.

    PubMed

    Salo, M K; Vartiainen, E; Puska, P; Nikkari, T

    1985-10-30

    Platelet aggregation and its relation to fatty acid composition of platelets, plasma and adipose tissue was determined in 196 randomly selected, free-living, 40-49-year-old men in two regions of Finland (east and southwest) with a nearly twofold difference in the IHD rate. There were no significant east-southwest differences in platelet aggregation induced with ADP, thrombin or epinephrine. ADP-induced platelet secondary aggregation showed significant negative associations with all C20-C22 omega 3-fatty acids in platelets (r = -0.26- -0.40) and with the platelet 20: 5 omega 3/20: 4 omega 6 and omega 3/omega 6 ratios, but significant positive correlations with the contents of 18:2 in adipose tissue (r = 0.20) and plasma triglycerides (TG) (r = 0.29). Epinephrine-induced aggregation correlated negatively with 20: 5 omega 3 in plasma cholesteryl esters (CE) (r = -0.23) and TG (r = -0.29), and positively with the total percentage of saturated fatty acids in platelets (r = 0.33), but had no significant correlations with any of the omega 6-fatty acids. Thrombin-induced aggregation correlated negatively with the omega 3/6 omega ratio in adipose tissue (r = -0.25) and the 20: 3 omega 6/20: 4 omega 6 ratio in plasma CE (r = -0.27) and free fatty acids (FFA) (r = -0.23), and positively with adipose tissue 18:2 (r = 0.23) and 20:4 omega 6 (r = 0.22) in plasma phospholipids (PL). The percentages of prostanoid precursors in platelet lipids, i.e. 20:3 omega 6, 20:4 omega 6 and 20:5 omega 3, correlated best with the same fatty acids in plasma CE (r = 0.32 - 0.77) and PL (r = 0.28 - 0.74). Platelet 20:5 omega 3 had highly significant negative correlations with the percentage of 18:2 in adipose tissue and all plasma lipid fractions (r = -0.35 - -0.44).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity.

    PubMed

    Smyth, Susan S; Sciorra, Vicki A; Sigal, Yury J; Pamuklar, Zehra; Wang, Zuncai; Xu, Yong; Prestwich, Glenn D; Morris, Andrew J

    2003-10-31

    Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production. PMID:12909631

  17. [Effect of soluble fibrin on the blood coagulation process and platelets aggregation].

    PubMed

    Zaichko, N V; Chernyshenko, T M; Platonova, T M; Volkov, H L

    2006-01-01

    The accumulation of soluble fibrin (SF) in the blood plasma causes acceleration of the final stage of blood coagulation. It increases functional activity of a hemostasis system platelet link, that is the precondition of thrombotic complication. Accumulation of SF in the blood plasma is accompanied by proportional reduction of coagulation time in ancistron and thrombin time tests, and also the intensification of platelets aggregation process. A conclusion was drawn that for early diagnostics of the DIC-syndrom it is expedient to carry out complex estimation of the hemostasis system with obligatory definition of the blood SF content, performance of ancistron and thrombin time tests, and also study of platelets aggregation.

  18. 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

  19. 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

  20. 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.

  1. Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation.

    PubMed

    Kuriyama, Shuhko; Kashiwagi, Hitoshi; Yuhki, Koh-ichi; Kojima, Fumiaki; Yamada, Takehiro; Fujino, Takayuki; Hara, Akiyoshi; Takayama, Koji; Maruyama, Takayuki; Yoshida, Akitoshi; Narumiya, Shuh; Ushikubi, Fumitaka

    2010-10-01

    The effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2-/- platelets) and EP4 (EP4-/- platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1-259 and AE1-329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2-/- and EP4-/- platelets. In human platelets, AE1-259 and AE1-329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1-329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1-259. AE1-329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3-208, an EP4 antagonist. In addition, AE1-329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

  2. 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

  3. Acute effect of a high-carbohydrate low-fat meal on platelet aggregation.

    PubMed

    Ahuja, Kiran D K; Adams, Murray J; Robertson, Iain K; Ball, Madeleine J

    2009-12-01

    Conflicting information is available regarding patient preparation with respect to the fasting and feeding states prior to blood collection in order to conduct platelet aggregation tests. Some literature suggests avoidance of only high-fat foods and allowance of non-fat foods and clear liquids; others suggest a fast of 8-10 hours. We conducted a study in 16 healthy subjects aged 44.0 +/- 12.7 (mean +/- SD) years to investigate and compare the effects of fasting and a high-carbohydrate low-fat meal on measures of platelet aggregation. Blood samples collected after an overnight fast of 10-12 hours and those collected at 40 and 120 minute postprandially (post-high-carbohydrate low-fat meal; 1900 kJ energy; 69, 16 and 15% of energy from carbohydrate, protein and fat, respectively), were tested for platelet aggregation in response to adenosine diphosphate. There was a significant reduction in maximum aggregation and area under the aggregation curve from fasting to 120 minute post meal (overall p < 0.001). Serum triglyceride concentrations did not change significantly from fasting to postprandial state (p = 0.53). Although there was a significant association between serum insulin, plasma glucose and measures of platelet aggregation, correcting for the effects of these metabolic parameters did not alter the results, providing evidence that other, currently unknown, factors associated with food consumption affect postprandial platelet aggregation. We propose that protocols for control of pre-analytical variables in platelet aggregation studies should make a fasting sample mandatory rather than "preferable" unless the objective of the study is to measure acute effects in response to a medication or food. PMID:19929247

  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. 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.

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

    PubMed Central

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

    2014-01-01

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

  8. 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

  9. The Effect of Ginger (Zingiber officinale) on Platelet Aggregation: A Systematic Literature Review

    PubMed Central

    Marx, Wolfgang; McKavanagh, Daniel; McCarthy, Alexandra L.; Bird, Robert; Ried, Karin; Chan, Alexandre; Isenring, Liz

    2015-01-01

    Background The potential effect of ginger on platelet aggregation is a widely-cited concern both within the published literature and to clinicians; however, there has been no systematic appraisal of the evidence to date. Methods Using the PRISMA guidelines, we systematically reviewed the results of clinical and observational trials regarding the effect of ginger on platelet aggregation in adults compared to either placebo or baseline data. Studies included in this review stipulated the independent variable was a ginger preparation or isolated ginger compound, and used measures of platelet aggregation as the primary outcome. Results Ten studies were included, comprising eight clinical trials and two observational studies. Of the eight clinical trials, four reported that ginger reduced platelet aggregation, while the remaining four reported no effect. The two observational studies also reported mixed findings. Discussion Many of the studies appraised for this review had moderate risks of bias. Methodology varied considerably between studies, notably the timeframe studied, dose of ginger used, and the characteristics of subjects recruited (e.g. healthy vs. patients with chronic diseases). Conclusion The evidence that ginger affects platelet aggregation and coagulation is equivocal and further study is needed to definitively address this question. PMID:26488162

  10. 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. PMID:27273565

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

    PubMed

    Peluso, Ilaria; Palmery, Maura; Serafini, Mauro

    2015-07-01

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

  12. The in vitro effect of aspirin on increased whole blood platelet aggregation in oral contraceptive users.

    PubMed

    Norris, L A; Bonnar, J

    1994-05-01

    The effects of triphasic oral contraceptives on whole blood platelet aggregation in 36 Italian women are reported here. Aspirin's effects on platelet aggregation were also studied. 18 women took a triphasic oral contraceptive; 10 women took Trinordiol, while 8 took Trinovum for at least 90 days. The remaining 18 women took nothing and served as controls. The study was aligned with each woman's birth control pill cycle. Blood was taken daily on days 15-21 of their cycle. Either saline solution or acetylsalicylic acid was added to the blood samples and compared. All data was statistically analyzed using unpaired student's t-test. Effects of 3 aggregating agents, ADP, PAF, and EDTA, on platelet aggregation were studied. Arachidonic acid and adrenalin bitartrate were also studied in this manner. An increase in platelet aggregation was observed in women taking oral contraceptives. No difference was found between patients taking Trinordiol and those taking Trinovum. The results of this study indicate an increase in whole blood platelet sensitivity to collagen, adrenalin, and arachidonic acid when using oral contraceptives. Aspirin, at low doses, may have a role in preventing early thrombus formation in women taking oral contraceptives. PMID:8042198

  13. Effects of Recombinant Human Megakaryocyte Growth and Development Factor (rHuMGDF) on Platelet Production, Platelet Aggregation, and Thrombosis.

    PubMed

    Lott; Nelson; Toombs

    1998-01-01

    Recombinant human megakaryocyte growth and development factor (rHuMGDF) is a c-mpl ligand that promotes the differentiation of CD34+ precursor cells into megakaryocyte, and then platelets. In experimental animals, injection of this and other c-mpl ligands leads to profound increases in the circulating platelet count in a matter of days. However, c-mpl ligands have also been shown to sensitize platelets to aggregating agents in vitro, raising the possibility that c-mpI ligands may have prothrombotic effects in vivo. Therefore, characterizing rHuMGDF in an in vivo model of thrombosis is a necessary and critical step in defining the in vivo pharmacology of this novel and important hernatopoietic factor, a pegylated form of which is currently in clinical trials. To determine the biologically effective doses in the rabbit, daily subcutaneous injections of rHuMGDF at 0.1, 1.0, or 10 µg/kg were administered ever 7 days. Daily injection of 10 µ/kg produced an approximate fourfold increase in platelet count and 1.0 µ/kg doubled platelet count over the injection period, both of which were statistically significant. The serum concentrations of rHuMGDF were determined 10 minutes following a single intravenous injection with 0.1, 1.0, and 10 µ/kg, and were 0.05 +/- 0.02, 0.98 +/- 0.07, and 21.32 +/- 21.35 ng/ml. To determine whether rHuMGDF can sensitize platelets in vivo, platelet aggregometry was performed on platelets isolated from animals immediately before and 10 minutes after they had been injected intravenously with rHuMGDF (0.1, 1.0, and 10 µ/kg). Intravenous injection of 10 µ/kg produced measurable changes in platelet aggregometry ex vivo, as evidenced by an increased sensitivity of platelets to adenosine diphosphate (ADP). To assess. the in vivo prothrombotic potential of rHuMGDF, a rabbit carotid artery model of cyclic flow reduction (CFR) was used to measure the effect of intravenous rHuMGDF administration on the rate of thrombus formation as assessed by CFR

  14. Effects of dihydropyridines and inorganic calcium blockers on aggregation and on intracellular free calcium in platelets.

    PubMed

    Palés, J; Palacios-Araus, L; López, A; Gual, A

    1991-05-01

    [Ca2+]i increase is necessary in physiological platelet activity, particularly aggregation and release. The increase of [Ca2+]i observed during platelet activation depends in part on Ca2+ influx from the extracellular medium. The participation of voltage-operated Ca2+ channels as a pathway for Ca2+ entry is controversial. In the present study we have attempted to reinvestigate this problem by measuring aggregation and [Ca2+]i changes in platelets activated by ADP or thrombin and incubated with organic or inorganic blockers of calcium channels. The main findings of the present paper can be summarized as follows: (i) Ni2+, Co2+ and Mn2+, well known inorganic blockers of Ca2+ channels, inhibited platelet aggregation induced by ADP or thrombin in a dose-dependent manner, Ni2+ being the most effective agent. (ii) Thrombin induced a rise in free [Ca2+]i in platelets incubated both in 1 mmol/l Ca(2+)-containing medium and in nominally Ca(2+)-free medium; the rise of free [Ca2+]i was in the first case up to 370 +/- 31 nmol/l and in the second case up to 242 +/- 26 nmol/l, indicating that this observed difference was due to Ca2+ entry from the extracellular medium. Co2+ and Ni2+ abolished that difference by inhibiting Ca2+ influx. (iii) Nisoldipine, nitrendipine and nimodipine (10-50 nmol/l) inhibited in a dose-dependent manner platelet aggregation induced by either ADP or thrombin in platelets incubated in normal-Ca2+ normal-K+ medium, also, aggregation was inhibited to a similar extent in platelets incubated in normal-Ca2+ high-K+ medium. (iv) Nisoldipine--the most effective dihydropyridine to inhibit platelet aggregation--also inhibited Ca2+ influx in platelets incubated in normal-Ca2+ medium, either in normal-K+ or high-K+ media. Our data support the existence of voltage-operated, dihydropyridine-sensitive calcium channels (L-type) and a physiological role for them in platelet function.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25601838

  16. 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.

  17. Inhibitory effect of compounds from Zingiberaceae species on human platelet aggregation.

    PubMed

    Jantan, I; Raweh, S M; Sirat, H M; Jamil, S; Mohd Yasin, Y H; Jalil, J; Jamal, J A

    2008-04-01

    Twelve compounds isolated from Alpinia mutica Roxb., Kaempferia rotunda Linn., Curcuma xanthorhiza Roxb., Curcuma aromatica Valeton and Zingiber zerumbet Smith (Family: Zingiberaceae) and three synthesized derivatives of xanthorrhizol were evaluated for their ability to inhibit arachidonic acid- (AA), collagen- and ADP-induced platelet aggregation in human whole blood. Antiplatelet activity of the compounds was measured in vitro by the Chrono Log whole blood aggregometer using an electrical impedance method. Among the compounds tested, curcumin from C. aromatica, cardamonin, pinocembrine and 5,6-dehydrokawain from A. mutica and 3-deacetylcrotepoxide from K. rotunda showed strong inhibition on platelet aggregation induced by AA with IC(50) values of less than 84 microM. Curcumin was the most effective antiplatelet compound as it inhibited AA-, collagen- and ADP-induced platelet aggregation with IC(50) values of 37.5, 60.9 and 45.7 microM, respectively.

  18. [Study on steroidal saponins from Dioscorea zingiberensis and their platelet aggregation activities].

    PubMed

    Wang, Jing-jing; Liu, Yi-xun; Wen, Di; Yu, He-shui; Kang, Li-ping; Pang, Xu; Zhao Yang; Ma, Bai-ping; Chen, Yun-dai

    2014-10-01

    Using the absorbent resin, silica gel and ODS column chromatography as well as semi-preparative HPLC, ten compounds were isolated from 70% ethanol extract of tubers of Dioscorea zingiberensis C. H. Wright, and their structures were elucidated as trigoneoside XIIIa (1), parvifloside (2), trigoneoside IVa (3), deltoside (4), protobioside (5), lilioglycoside k (6), zingiberensis newsaponin I (7), deltonin (8), prosapogenin A of dioscin (9), and trillin (10) on the basis of NMR and MS spectral data analysis. Among these compounds, 1, 3, 5 and 6 were isolated from this plant for the first time. In the screening test on platelet aggregation, compounds 7 and 8 exhibited induction effect on platelet aggregation, while compound 9 exhibited significant inhibitory effect on platelet aggregation in vitro.

  19. 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

  20. The Influence of Haemoglobin A1c Levels on Platelet Aggregation and Platelet Turnover in Patients with Coronary Artery Disease Treated with Aspirin

    PubMed Central

    Neergaard-Petersen, Søs; Hvas, Anne-Mette; Grove, Erik Lerkevang; Larsen, Sanne Bøjet; Gregersen, Søren; Kristensen, Steen Dalby

    2015-01-01

    Background Hyperglycaemia may attenuate the antiplatelet effect of aspirin and thereby increase the risk of cardiovascular events. We investigated the influence of increased haemoglobin A1c (HbA1c) levels on platelet aggregation and turnover in a large cohort of patients with coronary artery disease (CAD) with type 2 diabetes, prediabetes or no diabetes. Methods In this observational study, we included 865 stable CAD patients on 75 mg aspirin as mono-therapy of whom 242 patients had type 2 diabetes and were receiving antidiabetic drugs. Among 623 patients without diabetes, we classified 303 patients with prediabetes (HbA1c ≥5.7–6.4% [39–47 mmol/mol]) naive to antidiabetic drugs. Platelet aggregation was evaluated by the Multiplate Analyzer using arachidonic acid and collagen and by the VerifyNow Aspirin. Platelet turnover was evaluated by immature platelets using flow cytometry and platelet activation by soluble P-selectin. Results CAD patients with type 2 diabetes had higher platelet aggregation (all p-values <0.01), platelet turnover (immature platelet count, p<0.01) and platelet activation (p<0.001) than patients without diabetes. CAD patients with prediabetes had increased platelet aggregation (p = 0.02) and platelet count (p = 0.02) compared with patients without diabetes. Increased levels of HbA1c correlated positively with increased platelet aggregation using arachidonic acid (r = 0.19, p<0.0001), collagen (r = 0.10, p<0.01) and VerifyNow (r = 0.15, p<0.0001), and with platelet count (r = 0.08, p = 0.01), immature platelet count (r = 0.11, p<0.001) and soluble P-selectin (r = 0.15, p<0.0001). These associations were mainly evident in non-diabetic and prediabetic CAD patients. Conclusions CAD patients with prediabetes and diabetes may have attenuated antiplatelet effect of aspirin compared with CAD patients without diabetes. This may be related to increased platelet count in patients with prediabetes. Increased levels of HbA1c correlated positively

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

    PubMed

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

    2015-08-01

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

  2. Interference of IgG, IgG aggregates and immune complexes in tests for platelet autoantibodies.

    PubMed

    Helmerhorst, F M; Smeenk, R J; Hack, C E; Engelfriet, C P; von dem Borne, A E

    1983-11-01

    Three techniques, based on the antiglobulin principle, used for the detection of autoantibodies against platelets, were compared; the antiglobulin consumption assay (QACA), the platelet radioactive antiglobulin test (PRAT) and the platelet suspension immunofluorescence test (PSIFT). Upon incubation of normal donor platelets with purified IgG, in concentrations higher than that in serum, an increased amount of platelet-associated IgG was demonstrated only in the QACA. Upon incubation with aggregated IgG, all three tests became positive, but the PSIFT only with high concentrations of aggregates. Binding of soluble C1q-binding immune complexes (IC), which consisted of tetanus toxoid and IgG antitetanus antibodies (TaT) to normal donor platelets, was only detectable in the QACA. However, a positive result was obtained in all three tests with platelets incubated with soluble DNA-IgG-antiDNA antibodies (DaD) IC. Fixation of the platelets with paraformaldehyde prevented the binding and the detection of the DaD-IC, but not of IgG, aggregated IgG or TaT-IC. Eluates from platelets incubated with aggregated IgG, TaT- or DaD-IC did not react with normal donor platelets in the three techniques, in contrast to eluates from platelets sensitized with platelet antibodies.

  3. Inhibition of collagen-induced platelet aggregation by antibodies to distinct types of collagens.

    PubMed Central

    Balleisen, L; Nowack, H; Gay, S; Timpl, R

    1979-01-01

    Aggregation of platelets by fibrils formed from collagens type I, II and III could be inhibited by coating the fibrils with anti-collagen antibodies or Fab fragments. Similar results were obtained in a clot-retraction assay. Inhibition was achieved with stoichiometric amounts of antibodies and was specific for each type of collagen. Aggregation caused by a mixture of type-I and -III collagens could only be inhibited by a mixture of antibodies against both collagens. The data show that each interstitial collagen is capable of interacting with platelets and do not support the concept of an outstanding activity of type-III collagen. Images PLATE 1 PMID:395952

  4. 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

  5. Nafamostat mesilate, a broad spectrum protease inhibitor, modulates platelet, neutrophil and contact activation in simulated extracorporeal circulation.

    PubMed

    Sundaram, S; Gikakis, N; Hack, C E; Niewiarowski, S; Edmunds, L H; Koneti Rao, A; Sun, L; Cooper, S L; Colman, R W

    1996-01-01

    Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet beta-thromboglobulin (beta TG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-alpha 1-antitrypsin complex is 0.16 micrograms/ml in the NM group and 1.24 micrograms/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.

  6. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease. PMID:24219176

  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. Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity.

    PubMed

    Dizdarevic, Lili L; Biswas, Dipankar; Uddin, M D Main; Jørgenesen, Aud; Falch, Eva; Bastani, Nasser E; Duttaroy, Asim K

    2014-01-01

    Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit's cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease.

  9. Ticlopidine, Alka-Seltzer, or a combination of citric acid with aspirin: effects on platelet aggregation in individuals with an insufficient response to aspirin alone.

    PubMed

    Kaplan, S; Kaplan, A; Marcoe, K; Sauvage, L R

    2000-10-01

    Aspirin (ASA) does not effectively lower platelet aggregation in all people. The platelet aggregation (PA) score is an easily used clinical method for measuring the effect in individuals of antiplatelet medications. Fifteen apparently healthy subjects (2 men and 13 women), selected for their resistance to ASA's antiaggregation effect, completed a sequential trial of ticlopidine, Alka-Seltzer, and ASA + citric acid (CTA). Ticlopidine was the strongest aggregation inhibitor and the ASA + CTA combination was more inhibitory than Alka-Seltzer. It was determined that measuring antiaggregation effects of a particular agent in an individual prior to usage would optimize treatment. The PA score methodology provides a means for testing patients prior to antiplatelet therapy for prevention and treatment of the thrombotic complications of vascular disease. PMID:11030528

  10. Modulation of Platelet Activation and Thrombus Formation Using a Pan-PI3K Inhibitor S14161

    PubMed Central

    Ren, Lijie; Liu, Xiaohui; Wang, Qi; He, Sudan; Wu, Qingyu; Hu, Hu; Mao, Xinliang; Zhu, Li

    2014-01-01

    The phosphatidylinositol 3–kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. Flow cytometric studies showed that S14161 inhibited convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Comparable studies showed that S14161 has a higher potential bioavailability than LY294002, a prototypical inhibitor of pan-class I PI3K. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n = 9) compared to the vehicle controls (3.72±0.95 min, n = 8) (P<0.05), but did not prolong the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation without significant bleeding tendency and toxicity, and considering its potential higher bioavailability, it may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. PMID:25115838

  11. 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

  12. Ex vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect.

    PubMed

    Murayama, M

    1984-03-01

    Decompression of human platelet-rich plasma (PRP) in siliconized glass or plastic to 380 mm Hg for 3 hours at 38 degrees C produced platelet aggregation independent of pO2. Aggregation also took place when PRP was compressed to 8,000 PSI and then decompressed slowly to one atmosphere (14.7 PSI) without gas bubble formation. Platelets also aggregated when plasma was decompressed hydrodynamically (Bernoulli effect) at room temperature. It was also found that the drugs piracetam (2-oxypyrolidine acetamide) and pentoxifylline (1-(5-oxohexyl)-theobromine) at 0.5 and 1.0 mM prevent thrombocyte aggregation. Implications for mountain sickness are discussed.

  13. 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.

  14. New urushiols with platelet aggregation inhibitory activities from resin of Toxicodendron vernicifluum.

    PubMed

    Xie, Ya; Zhang, Jie; Liu, Wenyuan; Xie, Ning; Feng, Feng; Qu, Wei

    2016-07-01

    Eight new urushiol-type compounds (1-7b), along with seven known compounds were isolated from the resin of Toxicodendron vernicifluum Stokes. Their structures were determined by extensive spectroscopic methods, included (1)H NMR, (13)C NMR, HMQC, HMBC, HRESIMS, EI-MS in combination with CD methods. All the compounds except 7a and 7b were evaluated for their anti-platelet aggregation activities in vitro. Among them, compound 5 (IC50=5.12±0.85μmol/L), with a vic-diol moiety in the long alkyl chain showed the most potent inhibitory of platelet aggregation activity induced by ADP. In addition, compound 6 showed the effect of anti-platelet aggregation induced by AA with the IC50 value of 3.09±0.70μmol/L. Thus, these compounds might be the active components to the traditional use of Resina Toxicodendri for breaking up blood stasis, which could be related to the anti-platelet aggregation. PMID:27156871

  15. 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.

  16. Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets.

    PubMed

    Lim, H; Kubota, K; Kobayashi, A; Seki, T; Ariga, T

    1999-02-01

    The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane. PMID:10192909

  17. Platelets

    MedlinePlus

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

  18. 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

  19. 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. PMID:24833046

  20. [Anti-platelet aggregation bioassay based quality control for XST capsules].

    PubMed

    Han, Bing; Mao, Xin; Han, Shu-xian; Chen, Ying; Xiang, Yan-hua; Ge, Yi-meng; Liao, Fu-long; You, Yun

    2015-12-01

    A in vitro platelet aggregation bioassay was developed for the quality control of XST capsules. The in vitro anti-platelet aggregation effect in rats was observed to detect the bioactivity of XST capsules. Panax notoginseng saponins and Xuesaitong lyophilizedpowder for injection were taken as standard control substances to determine the potency. According to the results, XST capsules showeda significant inhibitory effect on thrombin-induced platelet aggregation in a dose-dependent manner. The in vitro anti-platelet activity oflyophilized powder for injection was stabler than that of Panax notoginseng saponins, and so suitable to serve as a standard control substance. The biological potency of XST capsules compared with standard control substance was detected by using parallel line assay. According to the results, the established bioassay method had a good repeatability (RSD 2.92%). The sample test results could pass thereliability test(linear deviation P > 0.05, parallel deviation P > 0.05). This bioassay method could be used as one of the complementary quality control methods for XST capsules.

  1. 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.

  2. 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. PMID:26910599

  3. Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

    PubMed Central

    Lombardi, Federica; Golla, Kalyan; Fitzpatrick, Darren J.; Casey, Fergal P.; Moran, Niamh; Shields, Denis C.

    2015-01-01

    Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling. PMID:25875950

  4. Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study.

    PubMed

    Scharbert, G; Kalb, M L; Essmeister, R; Kozek-Langenecker, S A

    2010-01-01

    The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30-34 degrees C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37 degrees C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate at five different test temperatures between 30 degrees C and 34 degrees C. Aggregation responses at 37 degrees C served as controls. At temperatures of mild and moderate hypothermia (30-34 degrees C), overall platelet aggregation was increased compared to 37 degrees C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31 degrees C and 34 degrees C and in response to adenosine diphosphate between 30 degrees C and 34 degrees C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30 degrees C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30 degrees C and 34 degrees C requires further detailed study. PMID:19954411

  5. An aggregation sensing reporter identifies leflunomide and teriflunomide as polyglutamine aggregate inhibitors

    PubMed Central

    Fuentealba, Rodrigo A.; Marasa, Jayne; Diamond, Marc I.; Piwnica-Worms, David; Weihl, Conrad C.

    2012-01-01

    Intracellular protein aggregation is a common pathologic feature in neurodegenerative diseases such as Huntington' disease, amyotrophic lateral sclerosis and Parkinson' disease. Although progress towards understanding protein aggregation in vitro has been made, little of this knowledge has translated to patient therapy. Moreover, mechanisms controlling aggregate formation and catabolism in cellulo remain poorly understood. One limitation is the lack of tools to quantitatively monitor protein aggregation and disaggregation. Here, we developed a protein-aggregation reporter that uses huntingtin exon 1 containing 72 glutamines fused to the N-terminal end of firefly luciferase (httQ72-Luc). httQ72-Luc fails to aggregate unless seeded by a non-luciferase-containing polyglutamine (polyQ) protein such as Q80-cfp. Upon co-aggregation, httQ72-luc becomes insoluble and loses its enzymatic activity. Using httQ72-Luc with Q80(CFP/YFP) as seeds, we screened the Johns Hopkins Clinical Compound Library and identified leflunomide, a dihydroorotate dehydrogenase inhibitor with immunosuppressive and anti-psoriatic activities, as a novel drug that prevents polyQ aggregation. Leflunomide and its active metabolite teriflunomide inhibited protein aggregation independently of their known role in pyrimidine biosynthesis, since neither uridine treatment nor other pyrimidine biosynthesis inhibitors affected polyQ aggregation. Inducible cell line and cycloheximide-chase experiments indicate that these drugs prevent incorporation of expanded polyQ into an aggregate. This study demonstrates the usefulness of luciferase-based protein aggregate reporters for high-throughput screening applications. As current trials are under-way for teriflunomide in the treatment of multiple sclerosis, we propose that this drug be considered a possible therapeutic agent for polyQ diseases. PMID:22052286

  6. 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.

  7. Upright posture and maximal exercise increase platelet aggregability and prostacyclin production in healthy male subjects

    PubMed Central

    Feng, D. L.; Murillo, J.; Jadhav, P.; McKenna, C.; Gebara, O. C.; Lipinska, I.; Muller, J. E.; Tofler, G. H.

    1999-01-01

    BACKGROUND: It is well accepted that heavy physical exertion can trigger the onset of myocardial infarction, but the mechanism is uncertain. As platelet and endothelial function play an important role in thrombotic events, platelet and prostacyclin responses to maximal treadmill exercise were studied. METHODS/RESULTS: The study subjects were 40 healthy men, mean (SEM) age 29 (5) years. Platelet aggregation was measured on a four channel aggregometer. Plasma 6-keto- prostaglandin F1alpha was analysed using an enzyme immunoassay technique. Upright posture and exercise produced an increase in platelet aggregability, as indicated by a fall in the threshold concentration of adrenaline (epinephrine) from 7.6 (1.5) microM at rest to 4.3 (1.0) microM after exercise (p = 0.002). The collagen lag time became significantly shorter with exercise (from 79.1 (3.1) seconds at rest to 71.9 (2.6) seconds after exercise, p = 0.003). Exercise was also associated with a 55% increase in plasma 6-keto-prostaglandin F1alpha (from 38.1 (75%CI 29.0 to 46.5) pg/ml at rest to 59.2 (47.3 to 66.8) pg/ml after exercise, p<0.001). CONCLUSIONS: In healthy male subjects, upright posture and maximal exercise increased platelet aggregability but this increase was counteracted by an increase in prostacyclin production. In patients with endothelial dysfunction, a reduced prostacyclin response to exercise may promote a transient prothrombotic imbalance that may trigger cardiovascular disease onset. 


 PMID:10597849

  8. 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.

  9. 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. PMID:26836594

  10. Obstruction of the lung capillaries by blood platelet aggregates and leucocytes in sudden infant death syndrome.

    PubMed

    Hanssen, Tor-Arne; Jørgensen, Leif

    2010-12-01

    Altogether 34 cases of sudden infant death were studied postmortem with particular emphasis on the pathological changes in the lungs. Light microscopy, including application of immunohistochemical methods, and transmission electron microscopy were used for the identification of blood platelets and white blood cell types in alveolar capillaries. The main findings were platelet aggregates and a varying number of neutrophil polymorphonuclear granulocytes in the lung capillaries, mixed with a smaller number of lymphocytes. The findings may be interpreted as an early sign of inflammation with capillary thrombosis, resulting in ischaemia, i.e. arrest of flow. In 21% of the cases, inflammatory cells had also expanded focally into alveolar spaces, creating the picture of localized areas of bronchopneumonia. An infant dying suddenly of a traumatic head injury served as a control. Neither platelets nor leucocytes were observed in the alveolar capillaries of this infant. In conclusion, in lungs from cases of sudden infant death syndrome, the alveolar capillaries are obstructed by platelet aggregates and leucocytes, interpreted as signs of an initial stage of lung inflammation with ischaemia. PMID:21091777

  11. The Constituents of Roots and Stems of Illigera luzonensis and Their Anti-Platelet Aggregation Effects

    PubMed Central

    Huang, Chieh-Hung; Chan, Yu-Yi; Kuo, Ping-Chung; Chen, Yu-Fon; Chang, Ren-Jie; Chen, Ih-Sheng; Wu, Shwu-Jen; Wu, Tian-Shung

    2014-01-01

    Phytochemical investigation of the roots and stems of Illigera luzonensis afforded two new aporphine alkaloids (1) and (2), one new bisdehydroaporphine alkaloid (3), and one new benzenoid (4), along with 28 known structures. The structures of new compounds were elucidated by spectral and MS analysis. Among the isolated compounds, (1) and (4–13) were subjected into the examination for their inhibitory effects on the aggregation of washed rabbit platelets. PMID:25089876

  12. Bis-spirolabdane diterpenoids from Leonurus japonicus and their anti-platelet aggregative activity.

    PubMed

    Xiong, Liang; Zhou, Qin-Mei; Peng, Cheng; Xie, Xiao-Fang; Liu, Lu-Si; Guo, Li; He, Ya-Cong; Yang, Lian; Liu, Zhao-Hua

    2015-01-01

    Six bis-spirolabdane diterpenoids along with four known analogues were isolated from the aerial parts of Leonurus japonicus. Their structures and absolute configurations were elucidated by spectroscopic analyses, single-crystal X-ray diffraction, and a modified Mosher's method. The inhibitory activity of the compounds against the abnormal increase in platelet aggregation induced by adenosine diphosphate was investigated. Only the (13R)-bis-spirolabdane diterpenoids exhibited a significant effect.

  13. 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

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

    PubMed

    Saunders, Janet C; Young, Lydia M; Mahood, Rachel A; Jackson, Matthew P; Revill, Charlotte H; Foster, Richard J; Smith, D Alastair; Ashcroft, Alison E; Brockwell, David J; Radford, Sheena E

    2016-02-01

    Protein aggregation underlies an array of human diseases, yet only one small-molecule therapeutic targeting this process has been successfully developed to date. Here, we introduce an in vivo system, based on a β-lactamase tripartite fusion construct, that is 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 islet amyloid polypeptide 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

  15. Novel β-amyloid aggregation inhibitors possessing a turn mimic.

    PubMed

    Hamada, Yoshio; Miyamoto, Naoko; Kiso, Yoshiaki

    2015-04-01

    Amyloid β peptide, the main component of senile plaques found in the brain of Alzheimer disease (AD) patients, is a molecular target for AD therapeutic intervention. A number of potential AD therapeutics have been reported, including inhibitors of β-secretase, γ-secretase, and Aβ aggregation, and anti-amyloid agents, such as neprilysin, insulin degrading enzyme (IDE), and Aβ antibodies. Recently, we reported potent small-sized β-secretase (BACE1) inhibitors, which could serve as anti-AD drugs. However AD is a progressive disorder, where dementia symptoms gradually worsen over several decades, and therefore may require many years to get cured. One possible way to achieve a greater therapeutic effect is through simultaneous administration of multiple drugs, similar to those used in Highly Active Anti-Retroviral Therapy (HAART) used to treat AIDS. In order to overcome AD, we took a drug discovery approach to evaluate, novel β-amyloid aggregation inhibitors. Previously, we reported that a tong-type compound possessing a turn mimic as the inhibitor of HIV-1 protease dimerization. Oligomerized amyloid β peptides contain a turn structure within the molecule. Here, we designed and synthesized novel β-amyloid aggregation inhibitors with a turn-mimic template, based on the turn conformer of the oligomerized amyloid β peptides.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

    Ryu, Si-Yun; Kim, Soochong

    2013-11-15

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

  18. Orally given gastroprotective capsaicin does not modify aspirin-induced platelet aggregation in healthy male volunteers (human phase I examination).

    PubMed

    Sandor, B; Papp, J; Mozsik, Gy; Szolcsanyi, J; Keszthelyi, Zs; Juricskay, I; Toth, K; Habon, Tamas

    2014-12-01

    Capsaicin is a well-known component of red pepper. Recent studies have shown that capsaicin could prevent gastric ulcer provoked by various NSAID-s like acetylsalicylic acid (ASA). Primary objective of this human clinical phase I trial was to investigate whether two different doses of capsaicin co-administered with ASA could alter the inhibitory effect of ASA on platelet aggregation. 15 healthy male subjects were involved in the study and treated orally with 400 μg capsaicin, 800 μg capsaicin, 500 mg ASA, 400 μg capsaicin+500 mg ASA and 800 μg capsaicin+500 mg ASA. Blood was drawn before and 1, 2, 6 and 24 hours after the drug administration. After that epinephrine induced platelet aggregation was measured by optical aggregometry. Between treatments, volunteers had a 6-day wash-out period. Our results showed that capsaicin had no effect on platelet aggregation, while as expected, ASA monotherapy resulted in a significant and clinically effective platelet aggregation inhibition (p ≤ 0.001). The combined ASA-capsaicin therapies reached equivalent effectiveness in platelet aggregation inhibition as ASA monotherapy. Our investigation proved that capsaicin did not influence the inhibitory effect of ASA on platelet aggregation, thus the capsaicin-ASA treatment would combine the antiplatelet effect of ASA with the possible gastroprotection of capsaicin.

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

    PubMed

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

    2014-07-01

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

  20. 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. PMID:27033179

  1. 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.

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

    PubMed

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

    2015-12-15

    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

  3. 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

  4. Dermcidin isoform-2 induced nullification of the effect of acetyl salicylic acid in platelet aggregation in acute myocardial infarction.

    PubMed

    Bank, Sarbashri; Jana, Pradipta; Maiti, Smarajit; Guha, Santanu; Sinha, A K

    2014-07-24

    The aggregation of platelets on the plaque rupture site on the coronary artery is reported to cause both acute coronary syndromes (ACS) and acute myocardial infarction (AMI). While the inhibition of platelet aggregation by acetyl salicylic acid was reported to produce beneficial effects in ACS, it failed to do in AMI. The concentration of a stress induced protein (dermcidin isoform-2) was much higher in AMI than that in ACS. Incubation of normal platelet rich plasma (PRP) with dermcidin showed one high affinity (Kd = 40 nM) and one low affinity binding sites (Kd = 333 nM). When normal PRP was incubated with 0.4 μM dermcidin, the platelets became resistant to the inhibitory effect of aspirin similar to that in the case of AMI. Incubation of PRP from AMI with dermcidin antibody restored the sensitivity of the platelets to the aspirin effect. Incubation of AMI PRP pretreated with 15 μM aspirin, a stimulator of the NO synthesis, resulted in the increased production of NO in the platelets that removed the bound dermcidin by 40% from the high affinity binding sites of AMI platelets. When the same AMI PRP was retreated with 10 μM aspirin, the aggregation of platelets was completely inhibited by NO synthesis.

  5. The use of quartz crystal microbalance with dissipation (QCM-D) for studying nanoparticle-induced platelet aggregation

    PubMed Central

    Santos-Martinez, Maria Jose; Inkielewicz-Stepniak, Iwona; Medina, Carlos; Rahme, Kamil; D’Arcy, Deirdre M; Fox, Daniel; Holmes, Justin D; Zhang, Hongzhou; Radomski, Marek Witold

    2012-01-01

    Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D) has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast and scanning helium ion microscopy. PMID:22275839

  6. 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. PMID:27534113

  7. 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.

  8. Effects of extracts and tannins from Arbutus unedo leaves on rat platelet aggregation.

    PubMed

    Mekhfi, Hassane; ElHaouari, Mohammed; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq

    2006-02-01

    Many cardiovascular diseases such as arterial hypertension are associated with an increase in blood platelet activity. Arbutus unedo (Ericaceae) is a medicinal plant reputed to treat arterial hypertension, so the present study was undertaken in order to determine the antiaggregant effect. The crude aqueous extract showed an inhibition of thrombin-induced platelet aggregation (IC50 = 1.8 +/- 0.09 g/L, n = 10). The subsequent extraction of Arbutus unedo leaves by successive solvents showed that the methanol and ethyl acetate extracts accounted for most of the antiaggregant activity (IC50 = 0.7 +/- 0.08, n = 9; 0.6 +/- 0.05; n = 9, respectively). The tannins isolated from the methanol extract exhibited a strong antiplatelet effect (% of inhibition = 75.3 +/- 1.4, n = 8) and may be the major chemical compounds responsible for this action. Our results support the traditional use of this plant in the preventive or therapeutic treatment of platelet aggregation linked to arterial hypertension. PMID:16444667

  9. Elucidation of flow-mediated tumour cell-induced platelet aggregation using an ultrasound standing wave trap

    PubMed Central

    Bazou, D; Santos-Martinez, MJ; Medina, C; Radomski, MW

    2011-01-01

    BACKGROUND AND PURPOSE Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood. EXPERIMENTAL APPROACH Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster–platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography. KEY RESULTS We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin. CONCLUSION AND IMPLICATIONS Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate–cancer cell clusters may be an important strategy to control metastasis. PMID:21182493

  10. 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.

  11. Collagen fibril aggregation-inhibitor from sea cucumber dermis.

    PubMed

    Trotter, J A; Lyons-Levy, G; Chino, K; Koob, T J; Keene, D R; Atkinson, M A

    1999-12-01

    Collagen fibrils from the dermis of the sea cucumber Cucumaria frondosa are aggregated in vitro by the dermal glycoprotein stiparin (Trotter et al., 1996). Under physiological ionic conditions stiparin appears to be both necessary and sufficient to cause fibrils to aggregate (Trotter et al., 1997). We report here the initial biochemical and biophysical characterization of a sulfated glycoprotein from C. frondosa dermis that binds stiparin and inhibits its fibril-aggregating activity. This inhibitory glycoprotein, which has been named 'stiparin-inhibitor,' has the highest negative charge density of all the macromolecules extracted from the dermis. SDS-PAGE reveals three approximately 31-kDa bands that stain with alcian blue but not with Coomassie blue. Analytical ultracentrifugation indicates a native molecular weight of 62 kDa. Transmission electron microscopy of rotary-shadowed molecules shows curved rods about 22 nm long. The glycoprotein does not bind collagen fibrils, but does bind stiparin with a 1:1 stoichiometry. The binding of stiparin-inhibitor to stiparin prevents the binding of stiparin to collagen fibrils. The carbohydrate moiety produced by papain-digestion of the glycoprotein retains all of its inhibitory activity. The carbohydrate moiety of the inhibitor is dominated by galactose and sulfate.

  12. Effects of tetrandrine and fangchinoline on experimental thrombosis in mice and human platelet aggregation.

    PubMed

    Kim, H S; Zhang, Y H; Yun, Y P

    1999-03-01

    Tetrandrine (TET) and fangchinoline (FAN) are two naturally occurring analogues with a bisbenzylisoquinoline structure. The present study was undertaken to investigate the effects of TET and FAN on the experimental thrombosis induced by collagen plus epinephrine (EP) in mice, and platelet aggregation and blood coagulation in vitro. In the in vivo study, the administration (50 mg/kg, i.p.) of TET and FAN in mice showed the inhibition of thrombosis by 55% and 35%, respectively, while acetylsalicylic acid (ASA, 50 mg/kg, i.p.), a positive control, showed only 30% inhibition. In the vitro human platelet aggregations induced by the agonists used in tests, TET and FAN showed the inhibitions dose dependently. In addition, neither TET nor FAN showed any anticoagulation activities in the measurement of the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using human-citrated plasma. These results suggest that antithrombosis of TET and FAN in mice may be mainly related to the antiplatelet aggregation activities. PMID:10193204

  13. Serotonin-induced platelet aggregation predicts the antihypertensive response to serotonin receptor antagonists.

    PubMed

    Gleerup, G; Persson, B; Hedner, T; Winther, K

    1993-01-01

    The 5-HT2-receptor antagonist ketanserin (20-40 mg b.i.d.) was administered to 62 patients of both sexes with uncomplicated primary hypertension. After 4 weeks of treatment about 50% of the patients had reached the target diastolic blood pressure of 90 mm Hg or below. Interindividual variability was large. In a retrospective analysis the variability could not be explained by sex or the dose of ketanserin. There was a weak association between age and systolic blood pressure response (r = 0.24; P = 0.06), which could be entirely accounted for by the higher base line blood pressure in the elderly patients. In one group of patients (n = 12), the ex vivo aggregation to serotonin (10(-6) M) was studied during treatment with placebo and ketanserin. Ketanserin completely inhibited 5-HT-induced aggregation in all patients. There was a close correlation between the area under the 5-HT-induced platelet aggregation curve during placebo and the subsequent reduction in diastolic blood pressure after 4 weeks of treatment with ketanserin. The present data suggest that the blood pressure response to ketanserin can be predicted from the ex vivo sensitivity of platelets to serotonin. By implication, they also support a role for serotonergic mechanisms in hypertension.

  14. Anti-CD31 delays platelet adhesion/aggregation at sites of endothelial injury in mouse cerebral arterioles.

    PubMed

    Rosenblum, W I; Murata, S; Nelson, G H; Werner, P K; Ranken, R; Harmon, R C

    1994-07-01

    The arterioles on the surface of the mouse brain (pial arterioles) were observed by in vivo microscopy. A focus of minor endothelial damage was produced in a single pial arteriole in each mouse by briefly exposing the site to a helium neon laser after an intravenous injection of Evans blue. Mice were injected 10 minutes before injury with a monoclonal antibody (MAb) to mouse CD31, also known as platelet endothelial cell adhesion molecule. This treatment doubled (P < .01) the time required for the laser to produce a recognizable platelet aggregate. In additional experiments, an MAb to mouse CD61 and an MAb to mouse intercellular adhesion molecule 1 had no effect. The data support previous observations indicating that platelet adhesion/aggregation in this model is induced by endothelial injury without exposure of basal lamina. The data are consistent with the hypothesis that the endothelial injury exposes or activates a platelet endothelial cell adhesion molecule on the endothelium which is blocked by the MAb directed against CD31. This may be the first demonstration of an effect of an anti-platelet endothelial cell adhesion molecule on platelet endothelial cell adhesion molecule on platelet adhesion/aggregation in vivo. PMID:8030753

  15. The impaired synthesis of insulin and its inability to inhibit platelet aggregation in cerebrovascular accident.

    PubMed

    Bank, Sarbashri; Bhattacharya, Suman; Maiti, Smarajit; Bhattacharya, Raja; Chakraborty, Debajyoti; Sinha, Asru K

    2015-10-01

    Both ischemic stroke (IS) and hemorrhagic stroke (HS) are reported to occur due to thrombosis on the arteries of the brain. As diabetes mellitus is a risk factor for strokes and insulin is reported to prevent thrombosis, the role of insulin in IS and HS was investigated. Forty eight stroke victims (IS = 22, HS = 26) and equal number of aged and sex matched normal volunteers participated in the study. Nitric oxide was determined by methemoglobin method. Insulin and Dermcidin isoform-2 (DCN2) level was determined by ELISA by using insulin and dermcidin antibody. Insulin binding to the platelet membrane was analyzed by scat chard plot. Treatment of normal platelet rich plasma (10(8)platelets/ml) with 15μUnits insulin/ml produced 1.41 nmol NO. The PRP from the IS and HS victims produced 0.38 nmol NO and 0.08 nmol NO respectively. Pretreatment of PRP from IS or HS subjects with 15 μM aspirin followed by 15μUnits of insulin/ml resensitized the platelets to the inhibitory effect of insulin. Mice hepatocytes treated with 0.14 μM DCN2 abolished the glucose induced insulin synthesis by NO that can be reversed by using 15 μM aspirin. It can be concluded that presence of DCN2 in stroke causes a condition similar to type I diabetes and nullified the effect of insulin in the inhibition of platelet aggregation in both IS and HS. The effect was reversed by 15 μM aspirin.

  16. Study of platelet aggregation in acute coronary syndrome with special reference to metabolic syndrome

    PubMed Central

    Paul, Rudrajit; Banerjee, Amit K; Guha, Shantanu; Chaudhuri, Utpal; Ghosh, Srabani; Mondal, Jayati; Bandyopadhyay, Ramtanu

    2013-01-01

    Background/Context: Antiplatelet drug resistance increases the risk of adverse events like stent thrombosis in acute coronary syndrome (ACS). Metabolic syndrome (MS) is a prothrombotic state and presence of MS further increases the risk of antiplatelet drug resistance. Aims and Objectives: We studied platelet aggregation characteristics in patients of ACS for aspirin or clopidogrel resistance. We studied the relation of drug resistance with blood markers like high sensitivity C-reactive protein (hsCRP). We also studied for any relation of drug resistance with presence of MS. Materials and Methods: We studied platelet aggregation characteristics by optical aggregometry using platelet-rich plasma (PRP) of patients. Collagen (2 μg/mL) and adenosine diphosphate (ADP; 10 μmol) were used. Greater than 50% aggregation in PRP of patients was taken as an evidence of drug resistance. Suitable blood tests were done including newer risk markers like hsCRP, apolipoprotein B, and fibrinogen. Statistical test: Statistical tests included Student's t-test and Kendall's rank correlation coefficient. Results: We had a total of 94 patients of ACS with 47 (50%) having MS. MS patients showed higher blood levels of hsCRP and fibrinogen. Twenty-eight (59.5%) patients with MS showed antiplatelet drug resistance compared to 12 patients without MS. Serum fibrinogen showed strongest correlation with drug resistance. HsCRP levels showed correlation with aspirin resistance (r = 0.53) only in the MS group. Discussion and Conclusion: We found significantly high prevalence of antiplatelet drug resistance. Aspirin and clopidogrel resistance was comparable. MS was a significant risk factor for drug resistance. The prothrombotic and proinflammatory markers showed strong correlation with drug resistance. A larger randomized trial is needed to better characterize this clinical problem. PMID:24083147

  17. Circulating platelet-leukocyte aggregates: a marker of microvascular injury in diabetic patients.

    PubMed

    Elalamy, I; Chakroun, T; Gerotziafas, G T; Petropoulou, A; Robert, F; Karroum, A; Elgrably, F; Samama, M-M; Hatmi, M

    2008-01-01

    Diabetes is associated with multiple disorders including metabolic, cellular and blood disturbances leading to vascular complications. Increased circulating levels of platelet-leukocyte aggregates (PLA) have been described in several thrombotic diseases. In this study, we have evaluated circulating PLA in diabetic patients and we have investigated whether they may be a marker of vascular complications. Using flow cytometry assay, we have quantified PLA percentages in 65 diabetics including 20 patients with type I and 45 with type II diabetes, and 25 healthy subjects. Specific labelling identified platelet-polymorphonuclear aggregates (PPA) and platelet-monocyte aggregates (PMA). We have observed a significant increase of PPA and PMA levels in diabetics (22+/-12% and 45+/-18%, respectively) compared to controls (7+/-4% and 19+/-10%, respectively) (p<0.01). However, both PPA and PMA values were similar in the two diabetes types. Circulating PPA and PMA were significantly enhanced in diabetics with vascular lesions (PPA: 24+/-13%; PMA: 50+/-18%) than in diabetics without vascular lesions (PPA: 18+/-8%; PMA: 38+/-15%) (p<0.05 and p<0.01). Patients with PPA>18% and/or PMA>38% showed a more important vascular injury (OR: 6; 95% CI: 1.6-23). Increased PMA circulating rate is particularly correlated to retinopathic injury (OR: 19; 95% CI: 2.3-154). Our findings established a relationship between increased circulating PLA levels, particularly PMA, and the incidence of microvascular complications in diabetes. They reinforce the concept of pro-inflammatory cells involvement in diabetic retinopathy pathogenesis and their link with thrombotic process. PMID:17825880

  18. 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).

  19. Structure-activity relationship of cyanine tau aggregation inhibitors

    PubMed Central

    Chang, Edward; Congdon, Erin E.; Honson, Nicolette S.; Duff, Karen E.; Kuret, Jeff

    2009-01-01

    A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3’-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity. PMID:19432420

  20. [Coumarins from Leonurus japonicus and their anti-platelet aggregative activity].

    PubMed

    Yang, Huai; Zhou, Qin-mei; Peng, Cheng; Liu, Lu-si; Xie, Xiao-fang; Xiong, Liang; Liu, Zhao-hua

    2014-11-01

    Chemical constituents of Leonurus japonicus were isolated and purified by a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, MCI, and Rp C18. Structures of the isolates were determined by spectroscopic analysis as 10 coumarins: bergapten (1), xanthotoxin (2), isopimpinellin (3), isogosferal (4), imperatorin (5), meransin hydrate(6), isomeranzin(7), murrayone(8) , auraptenol(9), and osthol(10). In addition to compound 9, the others were isolated from the genus Leonurus for the first time. In the in vitro assay, compounds 4 and 8 significantly inhibited the abnormal increase of platelet aggregation induced by ADP.

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

    SciTech Connect

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

    2014-07-04

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

  2. Roles of Mac-1 and glycoprotein IIb/IIIa integrins in leukocyte-platelet aggregate formation: stabilization by Mac-1 and inhibition by GpIIb/IIIa blockers.

    PubMed

    Patko, Zsofia; Csaszar, Albert; Acsady, Gyorgy; Peter, Karlheinz; Schwarz, Meike

    2012-01-01

    Circulating platelet-leukocyte hetero-aggregates play an important role in acute cardiovascular events and hypersensitivity reactions. The association involves the receptor families of selectins and integrin. The objective of this study was to investigate the role of CD11b/CD18 integrin (Mac-1) in hetero-aggregate formation and search for a counter-receptor on platelets ready to interact with Mac-1. As a model of leukocytes, Mac-1 presenting Chinese hamster ovary (CHO) cells were used to evaluate the role of Mac-1 in hetero-aggregate formation. The amount of CHO cell-bound active and inactive platelets was measured by flow cytometry, while the counter-receptors on platelets were identified via using blocking antibodies. We observed significant platelet adhesion on Mac-1-bearing cells when platelet-rich plasma or activated platelets were present. Inactive platelets did not adhere to Mac-1-bearing cells. Addition of fibrinogen, a ligand of Mac-1 significantly increased platelet binding. CD40L was demonstrated to act similarly on Mac-1. Inhibition of platelet GpIIb/IIIa completely abolished CHO cell-platelet aggregation. In our study, we have shown for the first time that Mac-1 mediates the formation of hetero-aggregates without selectin tethering when Mac-1 ligands such as fibrinogen or CD40L are present and blockers of platelet GpIIb/IIIa are able to diminish this interaction.

  3. The membrane potential modulates thrombin-stimulated Ca²⁺ mobilization and platelet aggregation.

    PubMed

    Albarrán, Letizia; Dionisio, Natalia; López, Esther; Salido, Ginés M; Rosado, Juan A

    2013-10-15

    G protein-coupled receptors can be directly modulated by changes in transmembrane voltage in a variety of cell types. Here we show that, while changes in the membrane voltage itself do not induce detectable modifications in the cytosolic Ca(2+) concentration, platelet stimulation with thrombin or the PAR-1 and PAR-4 agonist peptides SFLLRN and AYPGKF, respectively, results in Ca(2+) release from intracellular stores that is sensitive to the membrane depolarisation. Direct activation of G proteins or phospholipase C by AlF4(-) and m-3M3FBS, respectively, leads to Ca(2+) release that is insensitive to changes in the membrane potential. Thapsigargin-, as well as OAG-induced Ca(2+) entry are affected by the membrane voltage, probably as a result of the modification in the driving force for Ca(2+) influx; however, hyperpolarisation does not enhance thrombin- or OAG-evoked Ca(2+) entry probably revealing the presence of a voltage-sensitive regulatory mechanism. Transmembrane voltage also modulates the activity of the plasma membrane Ca(2+)-ATPase (PMCA) most likely due to a decrease in the phosphotyrosine content of the pump. Thrombin-stimulated platelet aggregation is modulated by membrane depolarisation by a mechanism that is, at least partially, independent of Ca(2+). These observations indicate that PAR-1 and PAR-4 receptors are modulated by the membrane voltage in human platelets. PMID:23988350

  4. Inhibitory effects of total flavones of Hippophae Rhamnoides L on thrombosis in mouse femoral artery and in vitro platelet aggregation.

    PubMed

    Cheng, Jiayi; Kondo, Kazunao; Suzuki, Yasuhiro; Ikeda, Yasuhiko; Meng, Xiansheng; Umemura, Kazuo

    2003-04-01

    Total flavones of Hippophae Rhamnoides L (TFH) are extracted from Sea buckthorn, a Chinese herbal medicine. Sea buckthorn has antioxidant, anti-ulcerogenic and hepato-protective actions, and its berry oil is reported to suppress platelet aggregation. Though it is frequently used for patients with thrombosis, the likely mechanism(s) and effects of TFH on thrombogenesis remain unclear. Thus, we have investigated the effect in-vivo of TFH on thrombogenesis and in vitro on platelet aggregation, comparing them to those of aspirin. We measured thrombotic occlusion time in a mouse femoral artery thrombosis model by the photochemical reaction between intravenously injected rose bengal and green light irradiation. In vitro platelet aggregation in whole blood was measured by single platelet counting. Thrombotic occlusion time was 8.5 +/- 0.6 min in the control group. TFH at a dose of 300 micro g/kg, intravenously administered 15 min before the rose bengal injection, significantly prolonged it to 11.6 +/- 1.0 min (P < 0.05), a similar effect on in-vivo thrombogenesis to that of aspirin. TFH at a concentration of 3.0 micro g/ml significantly (P < 0.01) inhibited in vitro platelet aggregation induced by collagen (2 micro g/ml) in a concentration dependent manner, in contrast TFH did not affect aggregation induced by arachidonic acid (80 micro M) and ADP (0.3 micro M). The results of the present study, in which TFH prevented in-vivo thrombogenesis, probably due to inhibition of platelet aggregation, suggest a possible clinical approach for the prevention of thrombosis. PMID:12628446

  5. 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

  6. Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

    NASA Astrophysics Data System (ADS)

    Wei, Yuxi; Wang, Changyun; Li, Jing; Guo, Qi; Qi, Hongtao

    2009-09-01

    We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA ( P<0.01) and increasing the synthesis of 6-keto-PGF1α, thus changing the plasma TXB2/6-keto-PGF1α balance when the platelets were activated ( P<0.01). Therefore, STP altered AA metabolism and these findings partly revealed the molecular mechanism by which STP inhibits ADP-induced platelet aggregation.

  7. Platelet aggregation responses and virus isolation from platelets in calves experimentally infected with type I or type II bovine viral diarrhea virus.

    PubMed

    Walz, P H; Bell, T G; Grooms, D L; Kaiser, L; Maes, R K; Baker, J C

    2001-10-01

    Altered platelet function has been reported in calves experimentally infected with type II bovine viral diarrhea virus (BVDV). The purpose of the present study was to further evaluate the ability of BVDV isolates to alter platelet function and to examine for the presence of a virus-platelet interaction during BVDV infection. Colostrum-deprived Holstein calves were obtained immediately after birth, housed in isolation, and assigned to 1 of 4 groups (1 control and 3 treatment groups). Control calves (n = 4) were sham inoculated, while calves in the infected groups (n = 4 for each group) were inoculated by intranasal instillation with 10(7) TCID50 of either BVDV 890 (type II), BVDV 7937 (type II), or BVDV TGAN (type I). Whole blood was collected prior to inoculation (day 0) and on days 4, 6, 8, 10, and 12 after inoculation for platelet function testing by optical aggregometry by using adenosine diphosphate and platelet activating factor. The maximum percentage aggregation and the slope of the aggregation curve decreased over time in BVDV-infected calves; however, statistically significant differences (Freidman repeated measures ANOVA on ranks, P < 0.05) were only observed in calves infected with the type II BVDV isolates. Bovine viral diarrhea virus was not isolated from control calves, but was isolated from all calves infected with both type II BVDV isolates from days 4 through 12 after inoculation. In calves infected with type I BVDV, virus was isolated from 1 of 4 calves on days 4 and 12 after inoculation and from all calves on days 6 and 8 after inoculation. Altered platelet function was observed in calves infected with both type II BVDV isolates, but was not observed in calves infected with type I BVDV. Altered platelet function may be important as a difference in virulence between type I and type II BVDV infection.

  8. Effects of a garlic-derived principle (ajoene) on aggregation and arachidonic acid metabolism in human blood platelets.

    PubMed

    Srivastava, K C; Tyagi, O D

    1993-08-01

    When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

  9. Effect of prenylated flavonoids and chalcones isolated from Artocarpus species on platelet aggregation in human whole blood.

    PubMed

    Jantan, Ibrahim; Mohd Yasin, Yusyila H; Jamil, Shajarahtunnur; Sirat, Hasnah; Basar, Norazah

    2010-07-01

    Five prenylflavonoids and two prenylchalcones from Artocarpus lowii King, A. scortechinii King and A. teysmanii Miq., and acetylated derivatives of cycloheterophyllin and artonin E were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP)-induced platelet aggregation in human whole blood by using an electrical impedance method. Among the tested compounds, only cycloheterophyllin inhibited AA-induced platelet aggregation with an IC(50) value of 100.9 microM. It also showed strong inhibition against ADP-induced aggregation, with an IC(50) value of 57.1 microM. Isobavachalcone, 2',4'-dihydroxy-4-methoxy-3'-prenyldihydrochalcone, cycloartobiloxanthone, artonin E and artonin E triacetate showed selective inhibition against ADP-induced aggregation, with IC(50) values ranging from 55.3 to 192.0 microM, but did not show such effect against other inducers.

  10. 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.

  11. 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

  12. Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

    PubMed Central

    Bonacina, F.; Barbieri, S.S.; Cutuli, L.; Amadio, P.; Doni, A.; Sironi, M.; Tartari, S.; Mantovani, A.; Bottazzi, B.; Garlanda, C.; Tremoli, E.; Catapano, A.L.; Norata, G.D.

    2016-01-01

    Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cell's derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis. PMID:26976330

  13. A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

    PubMed

    Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

    2016-08-01

    A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis.

  14. A novel thromboxane receptor antagonist, nstpbp5185, inhibits platelet aggregation and thrombus formation in animal models.

    PubMed

    Huang, Shiu-Wen; Kuo, Heng-Lan; Hsu, Ming-Tsung; Tseng, Yufeng Jane; Lin, Shu-Wha; Kuo, Sheng-Chu; Peng, Hui-Chin; Lien, Jin-Cherng; Huang, Tur-Fu

    2016-08-01

    A novel benzimidazole derivative, nstpbp5185, was discovered through in vitro and in vivo evaluations for antiplatelet activity. Thromaboxane receptor (TP) is important in vascular physiology, haemostasis and pathophysiological thrombosis. Nstpbp5185 concentration-dependently inhibited human platelet aggregation caused by collagen, arachidonic acid and U46619. Nstpbp5185 caused a right-shift of the concentration-response curve of U46619 and competitively inhibited the binding of 3H-SQ-29548 to TP receptor expressed on HEK-293 cells, with an IC50 of 0.1 µM, indicating that nstpbp5185 is a TP antagonist. In murine thrombosis models, nstpbp5185 significantly prolonged the latent period in triggering platelet plug formation in mesenteric and FeCl3-induced thrombi formation, and increased the survival rate in pulmonary embolism model with less bleeding than aspirin. This study suggests nstpbp5185, an orally selective anti-thrombotic agent, acting through blockade of TXA2 receptor, may be efficacious for prevention or treatment of pathologic thrombosis. PMID:27173725

  15. Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling.

    PubMed

    Su, Xing-li; Su, Wen; He, Zhi-long; Ming, Xin; Kong, Yi

    2015-09-01

    Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride-induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kβ with the binding free energy of -24.341 kcal/mol, which is close to that of cocrystallized ligand (-24.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases. PMID:25923322

  16. Tripeptide SQL Inhibits Platelet Aggregation and Thrombus Formation by Affecting PI3K/Akt Signaling.

    PubMed

    Su, Xing-li; Su, Wen; He, Zhi-long; Ming, Xin; Kong, Yi

    2015-09-01

    Centipede has been prescribed for the treatment of cardiovascular diseases in Asian countries for several hundred years. Previously, a new antiplatelet tripeptide SQL (H-Ser-Gln-Leu-OH) was isolated and characterized from centipede. In this study, we investigated its antithrombotic activities in vivo and underlying mechanism. It was found that SQL inhibited platelet aggregation induced by adenosine diphosphate, thrombin, epinephrine, and collagen and attenuated thrombus formation in both the ferric chloride-induced arterial thrombosis model and arteriovenous shunt thrombosis model in rats. It did not prolong the bleeding time in mice even at the dose of 10 mg/kg that showed potent antithrombosis effects. Molecular docking revealed that SQL binds PI3Kβ with the binding free energy of -24.341 kcal/mol, which is close to that of cocrystallized ligand (-24.220 kcal/mol). Additionally, SQL displayed inhibition on the late (180 seconds) but did not influence the early (60 seconds) Akt Ser473 phosphorylation in the immunoblot assay. These results suggest that SQL inhibits thrombus formation in vivo and that SQL inhibits PI3K-mediated signaling or even the PI3K itself in platelets. This study may help elucidate the mechanism for centipede treating cardiovascular diseases.

  17. Harpalycin 2 inhibits the enzymatic and platelet aggregation activities of PrTX-III, a D49 phospholipase A2 from Bothrops pirajai venom

    PubMed Central

    2012-01-01

    Background Harpalycin 2 (HP-2) is an isoflavone isolated from the leaves of Harpalyce brasiliana Benth., a snakeroot found in northeast region of Brazil and used in folk medicine to treat snakebite. Its leaves are said to be anti-inflammatory. Secretory phospholipases A2 are important toxins found in snake venom and are structurally related to those found in inflammatory conditions in mammals, as in arthritis and atherosclerosis, and for this reason can be valuable tools for searching new anti-phospholipase A2 drugs. Methods HP-2 and piratoxin-III (PrTX-III) were purified through chromatographic techniques. The effect of HP-2 in the enzymatic activity of PrTX-III was carried out using 4-nitro-3-octanoyloxy-benzoic acid as the substrate. PrTX-III induced platelet aggregation was inhibited by HP-2 when compared to aristolochic acid and p-bromophenacyl bromide (p-BPB). In an attempt to elucidate how HP-2 interacts with PrTX-III, mass spectrometry, circular dichroism and intrinsic fluorescence analysis were performed. Docking scores of the ligands (HP-2, aristolochic acid and p-BPB) using PrTX-III as target were also calculated. Results HP-2 inhibited the enzymatic activity of PrTX-III (IC50 11.34 ± 0.28 μg/mL) although it did not form a stable chemical complex in the active site, since mass spectrometry measurements showed no difference between native (13,837.34 Da) and HP-2 treated PrTX-III (13,856.12 Da). A structural analysis of PrTX-III after treatment with HP-2 showed a decrease in dimerization and a slight protein unfolding. In the platelet aggregation assay, HP-2 previously incubated with PrTX-III inhibited the aggregation when compared with untreated protein. PrTX-III chemical treated with aristolochic acid and p-BPB, two standard PLA2 inhibitors, showed low inhibitory effects when compared with the HP-2 treatment. Docking scores corroborated these results, showing higher affinity of HP-2 for the PrTX-III target (PDB code: 1GMZ) than aristolochic

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

    PubMed

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

    1989-05-01

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

  19. Pharmacokinetics of puerarin and ginsenoside Rg1 of CBN injection and the relation with platelet aggregation in rats.

    PubMed

    Liu, Ruining; Xing, Dongming; Lu, Hong; Wu, Hao; Du, Lijun

    2006-01-01

    In order to study the pharmacokinetics of puerarin and ginsenoside Rg1 of cerebral blood nutrition (CBN) and its relationship with pharmacodynamics of platelet aggregation induced by ADP in rat, the blood samples after injection were collected. The concentrations of puerarin and ginsenoside Rg1 in plasma were determined by RP-HPLC, and the platelet aggregations were observed simultaneously. The data showed that there was distinct statistic significance (p < 0.01) for puerarin processing, which was a single compartment model with quick elimination rate (t(1/2beta) = 18 min) and MRT (26 min), while ginsenoside Rg1 processing was a double compartment model with rapid distribution rate (t(1/2alpha) = 8 min), slow elimination rate (t(1/2beta) = 11 hours) and MRT (3.3 hours). Effects of anti-platelet aggregation were presented at 5-10 min, 45-90 min and 6-8 hours after injection separately, and the corresponding concentrations of puerarin were 25-21 microg/ml, 4.5-0.8 microg/ml and 0 microg/ml, ginsenoside Rg1 were 7.6-6.7 microg/ml, 1.2-0.6 microg/ml and 1.8-0.5 microg/ml. The two components presented a positive correlation between their concentrations and the effect of anti-platelet aggregation in 5-10 min after CBN injection (coefficient of correlation were 0.999 and 0.995). However, it was noted that the effect was still stronger even when concentrations of puerarin and ginsenoside Rg1 in plasma decreased. Therefore, puerarin and ginsenoside Rg1 not only had different pharmacokinetics, but also had a positive correlation with platelet aggregation, just in 5-10 min after CBN injection.

  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. ADP-induced platelet aggregation after addition of tramadol in vitro in fed and fasted horses plasma.

    PubMed

    Casella, S; Giannetto, C; Giudice, E; Marafioti, S; Fazio, F; Assenza, A; Piccione, G

    2013-04-01

    Adenosine diphosphate (ADP)-induced platelet aggregation in fed and fasted horses after addition of tramadol hydrochloride was evaluated in vitro. On 10 horses citrated blood samples were collected 2h after feeding (fed animals) and 21 h after feeding (fasted animals). Final concentrations of ADP 1 and 0.5 μM, and tramadol hydrochloride (1, 15, 30, 45 and 60 min after the addition of tramadol) were used to determine the maximum degree and initial velocity of platelet aggregation. Repeated measures multifactor analysis of variance (MANOVA) was used to evaluate the effect of feeding/fasting condition, ADP concentration and addition of tramadol. Findings showed statistical differences (P≤0.05) on studied parameters after addition of tramadol to different ADP concentrations in fed and fasted horses. The clinical relevance of these results is that tramadol provides many advantages as a therapeutic option; in fact, it is an inexpensive and a relatively new analgesic in equine veterinary medicine. Further investigations would be appropriate to compare the effects of different opioids but also using different concentrations of tramadol associated with other drugs in order to have substances which can regulate the functional activity of the platelets and to extend the knowledges on equine platelet aggregation. PMID:23031839

  3. 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

  4. 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.

  5. 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. PMID:26406321

  6. 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

  7. 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

  8. Antithrombotic effects of losartan in patients with hypertension complicated by atrial fibrillation: 4A (Angiotensin II Antagonist of platelet Aggregation in patients with Atrial fibrillation), a pilot study.

    PubMed

    Sakamoto, Tomohiro; Kudoh, Takashi; Sakamoto, Kenji; Matsui, Kunihiko; Ogawa, Hisao

    2014-06-01

    Angiotensin receptor blockers (ARBs) are widely used for the treatment of hypertension. It has been reported that the ARB losartan has antiplatelet, anticoagulant and profibrinolytic effects experimentally. These properties could be desirable to treat hypertensive patients with high atherothrombotic and/or thromboembolic risk. To examine the antithrombotic effects of losartan in hypertension, 20 consecutive patients with hypertension complicated by atrial fibrillation (AF) were enrolled in this study. The patients were treated with losartan 50 mg for 8 weeks followed by 100 mg for 4 weeks. Blood samples were obtained from each patient at 0 (pretreatment), 8 and 12 weeks after initiating treatment. Platelet aggregability, plasma levels of tissue factor (TF) and type 1 plasminogen activator inhibitor (PAI-1) activity levels were measured. The area under the curve for small platelet aggregability decreased from 100 to 42.8% at 12 weeks (P<0.0001). TF levels (ng ml(-1)) and PAI-1 activity (IU ml(-1); mean±s.d.) also changed from 14.2±3.6 to 10.9±4.5 at 12 weeks (P=0.0299) and from 11.7±3.6 to 8.5±3.1 at 12 weeks (P=0.0122), respectively. Losartan inhibited platelet activity and coagulation factors in a dose- and time-dependent manner in patients with hypertension complicated by AF, whereas the fibrinolytic capacity was increased. The use of losartan could be advantageous in the treatment of hypertensive patients with high atherothrombotic risk.

  9. 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

  10. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes: III. Shear and extrinsic fibrinogen-dependent effects.

    PubMed

    Goldsmith, H L; Frojmovic, M M; Braovac, S; McIntosh, F; Wong, T

    1994-01-01

    The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23 degrees C was studied using a previously described double infusion technique and resistive particle counter size analysis. Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 x 10(5) microliters-1; (17)] with [fibrinogen] from 0 to 1.2 microM, the rate and extent of aggregation with 0.7 microM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, G, = 41.9, 335 and 1,335 s-1. As measured by the decrease in singlet concentration, aggregation at 1.2 microM fibrinogen increased with increasing G up to 1,335 s-1, in contrast to that previously reported in citrated plasma, in which aggregation reached a maximum at G = 335 s-1. Without added fibrinogen, there was no aggregation at G = 41.9 s-1; at G = 335 s-1, there was significant aggregation but with an initial lag time, aggregation increasing further at G = 1,335 s-1. Without added fibrinogen, aggregation was abolished at all G upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab')2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37 degrees C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab')2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of

  11. 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

  12. 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.

  13. Eugenol: a dual inhibitor of platelet-activating factor and arachidonic acid metabolism.

    PubMed

    Saeed, S A; Simjee, R U; Shamim, G; Gilani, A H

    1995-07-01

    Eugenol is an active principal and responsible for several pharmacological activities of clove oil. We studied the effects of eugenol on human platelet aggregation, arachidonic acid (AA) and platelet-activating factor (PAF) metabolism and in vivo effects on AA and PAF-induced shock in rabbits. Eugenol strongly inhibited PAF-induced platelet aggregation with lesser effect against AA and collegen. The IC(50) values were against AA: 31 ± 0.5; collagen: 64 ± 0.7 and PAF 7 ± 0.2 μM (n=9) respectively. In addition, eugenol stimulated PAF-acetylhydrolase activity suggesting that inhibition of PAF could be due to its inactivation to lyso-PAF. Pretreatment of rabbits with eugenol (50-100 mg/kg) prevented the lethal effects of intravenous PAF (11 μgg/kg) or AA (2 mg/kg) in a dose-dependent fashion. The protective effects of eugenol in the rabbits, however, were more pronounced against PAF-induced mortality (100% protection). In addition, eugenol also inhibited AA metabolism via cyclooxygenase and lipoxygenase pathways in human platelets. Both the production of thromboxane-A(2) and 12-hydroxy-eicosatetraenoic acid was inhibited by eugenol in a concentration-related manner (30-120 μM). In vivo, eugenol (50-100 mg/kg; i.p.) inhibited carrageenan-induced rat paw oedema (P < 0.001). In this test, eugenol was 5 times more potent than aspirin. These results provide evidence that eugenol acts as a dual antagonist of AA and PAF. PMID:23196096

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

    PubMed

    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).

  15. [Palm oil derivatives with different concentration of palmitic acid and antioxidants. Effects upon plasmatic lipids and platelet aggregation].

    PubMed

    Scorza, T; Martucci, A; Torrealba de Ron, A T

    1999-03-01

    It was evaluated the effect of diet rich with cholesterol (0.1%) and different concentration of palmitic acid (16:0) and antioxidants (vitamin C, alpha tocopherol and retinol) upon plasmatic lipids and platelet aggregability in rabbits. The animals were distributed in three groups: I. Standard chow meal (Rp Conejarina) + cholesterol (chol) 0.1%; II. Standard chow meal + chol 0.1% + semipurified palm oil 10% (16:0 = 39.8%, oleic acid 48.7%, linoleic acid 11.4%, retinol 7.3 ug/dL, alpha tocopherol 157.6 ug/dL; III. Standard chow meal + chol 0.1% + crude palm oil 10% (16:0 = 45.3%, oleic acid 46.3%, linoleic acid 7.9%, retinol 96.4 ug/dL, alpha tocopherol 322.8 ug/dL). Monthly determination of plasmatic lipids were done (Enzymatic methods) and at ten months platelet aggregability with ADP, plasmatic vitamin C, retinol and, alpha tocopherol determination were done. Total plasmatic cholesterol (TC) and LDLc increased significantly in the three groups of animals. Significant differences between groups were not found. Platelet aggregability was lower in the animals fed with palmitic acid rich diet (groups II and III) (P = 0.002 and 0.001). Retinol, alpha tocopherol plasmatic concentrations revealed no significant differences. Vitamin C in the groups I was lower than groups II and III (P < 0.05 < 0.02). In this study hypercholesterolemic rabbits fed with rich diets (crude and semipurified) had lower platelet aggregability without changes in plasmatic lipids concentrations.

  16. 2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation.

    PubMed

    Cristalli, G; Volpini, R; Vittori, S; Camaioni, E; Monopoli, A; Conti, A; Dionisotti, S; Zocchi, C; Ongini, E

    1994-05-27

    A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.

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

    PubMed

    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

  18. The effects of an inhibitor of diglyceride lipase on collagen-induced platelet activation.

    PubMed

    Jackson, Elke C G; Ortar, Giorgio; McNicol, Archie

    2013-12-01

    Human platelet activation by collagen occurs in a dose-dependent manner. High concentrations of collagen bind to a pair of receptors, the α2β1 integrin and glycoprotein (GP)VI/Fc-receptor γ-chain (FcRγ), which stimulate a cascade of events including Syk, LAT, Btk, Gads, and phospholipase Cγ2, leading to calcium release and protein kinase C (PKC) activation. Calcium and PKC are responsible for a range of platelet responses including exocytosis and aggregation, as well as the cytosolic phospholipase A2 (cPLA2)-mediated release of arachidonic acid, which is converted to thromboxane (Tx)A2. In contrast, low concentrations of collagen are acutely aspirin-sensitive, and calcium release and aggregation are TxA2-dependent. Under these conditions, cPLA2 is not involved and it has been suggested that phospholipase C generates 1,2-diacylglycerol (DG) from which arachidonic acid is liberated by diglyceride lipase (DGL). Here a novel DGL blocker (OMDM-188) inhibited collagen-, but not arachidonic acid-induced aggregation and TxA2 synthesis. Furthermore, OMDM-188 inhibited collagen-induced arachidonic acid release. Finally OMDM-188 inhibited collagen-induced p38(MAPK) phosphorylation, but not extracellular signal-regulated kinase (ERK) phosphorylation, with no effect on the phosphorylation of either enzyme in response to arachidonic acid. Taken together, these data suggest a role for a pathway involving phospholipase C liberating DG from membrane phospholipids in response to minimally activating concentrations of collagen. The DG serves as a substrate for DGL, potentially under the regulations of p38(MAPK), to release arachidonic acid, which is subsequently converted to TxA2, which mediates the final platelet response.

  19. Osmotic stability of blood platelets

    PubMed Central

    Fantl, P.

    1968-01-01

    1. Hypotonic solutions added to human platelet-containing plasma cause a transient decrease of absorbancy of light at 610 mμ which is followed by a gradual increase of absorbancy. 2. When platelets are stored for 7 hr at 4° C the absorbancy changes with variations of osmolarity and their aggregation with adenosine diphosphate (ADP) remain the same. However, the reversal of absorbancy declines during storage of platelet-containing plasma. 3. Platelets are not aggregated by stearate. Platelets appear to be only slightly affected by stearate concentration higher than 0·8 mM, but oleate has no effect. 4. Hypertonic solutions of NaCl and urea cause increase in absorbancy of platelet-containing human plasma. Hypertonic sucrose solutions produce no more change than isotonic solutions. Hypertonic NaCl produces permanent increases in absorbancy. In human platelet-containing plasma the increased absorbancy caused by hypertonic urea is transient and declines. 5. The osmotic platelet changes occur in isolated platelets as well as in platelet-containing plasma. 6. The absorbancy of frozen and thawed platelet-containing plasma is not significantly altered by hypotonic solutions but the absorbancy changes caused by hypertonic solutions are similar to that of unfrozen plasma. 7. The immediate absorbancy changes caused by hypo- and by hypertonic solutions are the same at 5° C and 30° C and are therefore probably of a physical nature. The reversal of absorbancy and aggregation of platelets by added adenosine diphosphate have Q10 > 1 and are therefore probably of a chemical-enzymic nature. 8. Divalent cations and contact activation are not required for the osmotic platelet changes and 10-3 M-Cu2+ and Zn2+ do not interfere. Inhibitors of oxidative phosphorylation, electron transfer, sodium, potassium activated adenosine triphosphatases and adenosine triphosphate do not inhibit reversal of absorbancy of platelets exposed to hypotonic solutions. Cyanide, 5 × 10-3 M, fluoride, 1

  20. Inhibition of the effects of thrombin on guinea pig platelets by the diacylglycerol lipase inhibitor RHC 80267

    SciTech Connect

    Amin, D.; Sutherland, C.A.; Khandwala, A.S.; Jamall, I.S.; Kapoor, A.L.

    1986-10-01

    Phospholipase C (PLC) and diacylglycerol lipase (DGL) activities were found in guinea pig platelet microsome preparations. No phospholipase A2 (PLA2) activity was detected. RHC 80267 (1,6-di (0-(carbamoyl) cyclohexanone oxime)hexane) inhibited DGL activity (IC50 = 4 uM) from guinea pig platelet microsomes but had no effect on PLC. RHC 80267 inhibited platelet aggregation (IC50 = 11 uM), release of arachidonic acid (AA), its metabolites, and ATP (IC50 = 4.5 uM) when guinea pig platelets were challenged with a low concentration of thrombin. We propose that PLC-DGL is an important enzymatic pathway for the release of AA in guinea pig platelets.

  1. Effect of Vitamin C Supplementation on Platelet Aggregation and Serum Electrolytes Levels in Streptozotocin-Induced Diabetes Mellitus in Rats.

    PubMed

    Owu, Daniel U; Nwokocha, Chukwuemeka R; Ikpi, Daniel E; Ogar, Emmanuel I

    2016-01-01

    Diabetes mellitus (DM) is a disease condition characterised by hyperglycemia; free radical and abnormalhaematological indices. Vitamin C can reduce free radical generation and ameliorate adverse conditions of diabetes mellitus.The aim of the present study is to investigate the effect of vitamin C on platelet aggregation and electrolyte levels in Type 1DM. Male Wistar rats were divided into four groups namely control, DM, DM +Vitamin C and Vitamin C groups. Rats weremade diabetic with a single dose of streptozotocin (65 mg/kg) intraperitoneally. Vitamin C was administered orally todiabetic and normal rats at 200 mg/kg body weight for 28 days. Blood samples were analyzed for hematological parameters,platelet aggregation, and serum electrolyte levels. Blood glucose in DM+ Vitamin C group (9.9 ± 1.8 mmol/L) wassignificantly reduced (p<0.01) compared to DM group (32.2 ± 2.1 mmol/L) and significantly higher (p<0.05) than control(4.4 ± 0.8 mmol/L). Haemoglobin (Hb) concentration in DM group (12 ± 0.1 g/dL) was significantly reduced (p<0.01) whencompared with control groups (14 ± 0.24 g/dL) and significantly increased (p<0.05) in the DM+vitamin C group (13.5 ± 0.5g/dL) compared with the diabetic group. The mean corpuscular volume values in DM (68.66 ± 0.5 fL) and DM+vitamin Cgroups (68.11 ± 0.4 fL) were significantly higher (p<0.01) than the control (59.49 ± 0.5fL). Platelet count in DM group (523± 8.5 x109/L) was significantly raised (p<0.01) when compared to control (356 ± 6.2 x109/L) and significantly reduced(p<0.01) in DM+ vitamin C-treated group (385 ± 7.8 x109/L) compared with DM group. Platelet aggregation and serumsodium/potassium ratios was significantly reduced (p<0.01) in DM+vitamin C compared with DM group. These resultssuggest that oral vitamin C administration increases haemoglobin, reduced plasma glucose level, platelet count, serumsodium/potassium ion ratio and inhibits platelet aggregation in streptozotocin-induced DM in rats. PMID:27574765

  2. Parnaparin, a low-molecular-weight heparin, prevents P-selectin-dependent formation of platelet-leukocyte aggregates in human whole blood.

    PubMed

    Maugeri, Norma; Di Fabio, Giovannina; Barbanti, Miriam; de Gaetano, Giovanni; Donati, Maria Benedetta; Cerletti, Chiara

    2007-06-01

    Parnaparin, a low-molecular-weight heparin (LMWH), prevents platelet activation and interaction with polymorphonuclear leukocyte (PMN) in a washed cell system. The in-vitro effect of parnaparin was studied here on platelet-PMN aggregates formed with more physiologic approaches in whole blood, in parallel with unfractionated heparin and enoxaparin, another LMWH. Citrated blood from healthy subjects was stimulated: i) from passage through the "Platelet Function Analyzer" (PFA-100), a device that exposes blood to standardized high shear flow through collagen/ADP cartridges; ii) by collagen and ADP (2 and 50 mug/ml, respectively) added in combination under stirring in an aggregometer cuvette; iii) with recombinant Tissue Factor, to generate thrombin concentrations able to activate platelets without inducing blood clotting, or iv) the Thrombin Receptor Activating Peptide-6 (TRAP-6). Platelet P-selectin and platelet-PMN aggregates were measured by flow cytometry upon stimulation of blood. Fibrinogen binding to platelets and markers of PMN activation were also detected. Platelet P-selectin expression and platelet-PMN aggregate formation were induced in all four activation conditions tested. Parnaparin prevented in a concentration-dependent manner (0.3-0.8 IUaXa/ml) the expression of P-selectin and the formation of mixed aggregates, while the two reference heparin preparations had a much weaker effect. Platelet fibrinogen binding and PMN activation markers (fibrinogen binding, CD11b and CD40) were also prevented by parnaparin. These data extend in more physiological systems of platelet activation, the anti-inflammatory profile of parnaparin, previously reported in washed cells. The greater effect of parnaparin, as compared to the reference heparins, could be due to chemico-physical differences possibly unrelated to their anticoagulant effect. PMID:17549299

  3. Differences in Whole Blood Platelet Aggregation at Baseline and in Response to Aspirin and Aspirin Plus Clopidogrel in Patients With Versus Without Chronic Kidney Disease.

    PubMed

    Jain, Nishank; Li, Xilong; Adams-Huet, Beverley; Sarode, Ravi; Toto, Robert D; Banerjee, Subhash; Hedayati, S Susan

    2016-02-15

    Thrombotic events while receiving antiplatelet agents (APAs) are more common in subjects with versus without chronic kidney disease (CKD). Data on antiplatelet effects of APA in CKD are scarce and limited by lack of baseline platelet function before APA treatment. We hypothesized subjects with stages 4 to 5 CKD versus no CKD have greater baseline platelet aggregability and respond poorly to aspirin and clopidogrel. In a prospective controlled study, we measured whole blood platelet aggregation (WBPA) in 28 CKD and 16 non-CKD asymptomatic stable outpatients not on APA, frequency-matched for age, gender, obesity, and diabetes mellitus. WBPA was remeasured after 2 weeks of each aspirin and aspirin plus clopidogrel. The primary outcome was percent inhibition of platelet aggregation (IPA) from baseline. The secondary outcome was residual platelet aggregability (RPA; proportion with <50% IPA). Baseline platelet aggregability was similar between groups except adenosine diphosphate-induced WBPA, which was higher in CKD versus non-CKD; median (interquartile range) = 13.5 (9.5 to 16.0) versus 9.0 (6.0 to 12.0) Ω, p = 0.007. CKD versus non-CKD participants had lower clopidogrel-induced IPA, 38% versus 72%, p = 0.04. A greater proportion of CKD versus non-CKD participants had RPA after clopidogrel treatment (56% vs 8.3%, p = 0.01). There were no significant interactions between CKD and the presence of cytochrome P450 2C19 polymorphisms for platelet aggregability in clopidogrel-treated participants. In conclusion, CKD versus non-CKD subjects exhibited similar platelet aggregation at baseline, similar aspirin effects and greater RPA on clopidogrel, which was independent of cytochrome P450 2C19 polymorphisms. PMID:26725101

  4. Differences in Whole Blood Platelet Aggregation at Baseline and in Response to Aspirin and Aspirin Plus Clopidogrel in Patients With Versus Without Chronic Kidney Disease.

    PubMed

    Jain, Nishank; Li, Xilong; Adams-Huet, Beverley; Sarode, Ravi; Toto, Robert D; Banerjee, Subhash; Hedayati, S Susan

    2016-02-15

    Thrombotic events while receiving antiplatelet agents (APAs) are more common in subjects with versus without chronic kidney disease (CKD). Data on antiplatelet effects of APA in CKD are scarce and limited by lack of baseline platelet function before APA treatment. We hypothesized subjects with stages 4 to 5 CKD versus no CKD have greater baseline platelet aggregability and respond poorly to aspirin and clopidogrel. In a prospective controlled study, we measured whole blood platelet aggregation (WBPA) in 28 CKD and 16 non-CKD asymptomatic stable outpatients not on APA, frequency-matched for age, gender, obesity, and diabetes mellitus. WBPA was remeasured after 2 weeks of each aspirin and aspirin plus clopidogrel. The primary outcome was percent inhibition of platelet aggregation (IPA) from baseline. The secondary outcome was residual platelet aggregability (RPA; proportion with <50% IPA). Baseline platelet aggregability was similar between groups except adenosine diphosphate-induced WBPA, which was higher in CKD versus non-CKD; median (interquartile range) = 13.5 (9.5 to 16.0) versus 9.0 (6.0 to 12.0) Ω, p = 0.007. CKD versus non-CKD participants had lower clopidogrel-induced IPA, 38% versus 72%, p = 0.04. A greater proportion of CKD versus non-CKD participants had RPA after clopidogrel treatment (56% vs 8.3%, p = 0.01). There were no significant interactions between CKD and the presence of cytochrome P450 2C19 polymorphisms for platelet aggregability in clopidogrel-treated participants. In conclusion, CKD versus non-CKD subjects exhibited similar platelet aggregation at baseline, similar aspirin effects and greater RPA on clopidogrel, which was independent of cytochrome P450 2C19 polymorphisms.

  5. Synthesis of analogues of gingerol and shogaol, the active pungent principles from the rhizomes of Zingiber officinale and evaluation of their anti-platelet aggregation effects.

    PubMed

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

    2014-01-01

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

  6. A monoclonal antibody directed against a granule membrane glycoprotein (GMP-140/PADGEM, P-selectin, CD62P) inhibits ristocetin-induced platelet aggregation.

    PubMed

    Boukerche, H; Ruchaud-Sparagano, M H; Rouen, C; Brochier, J; Kaplan, C; McGregor, J L

    1996-02-01

    P-selectin (also called CD62, GMP-140, PADGEM, CD62P) is a recently described member of a family of vascular adhesion receptors expressed by activated platelets and endothelial cells that are involved in leucocyte cell adhesion. The aim of this study was to characterize a new monoclonal antibody (LYP7) directed against activated human blood platelets that inhibits ristocetin-induced platelet aggregation. Immunoadsorbent affinity chromatography and immunoprecipitation studies showed that LYP7 (IgG1) bound a surface-labelled glycoprotein (GP) which changed its apparent molecular mass (M(r)) on reduction from 138 kD (situated below GPIIb) to 148 kD (above GPIIb alpha). LYP7 and S12, a monoclonal antibody directed against P-selectin immunoprecipitated the same band. Using ELISA assay, purified P-selectin was shown to bind LYP7 and S12 monoclonal antibodies. Binding sites of 125I-labelled LYP7, which was greatly increased on thrombin-stimulated (2 U/ml) washed platelets (10825 +/- 2886, mean +/- SD) Kd = 1.5 +/- 0.5 nM) compared to resting platelets (2801 +/- 1278, mean +/- SD) (Kd = 1.5 +/- 0.6 nM), was found to be normal on thrombin-stimulated platelets taken from a patient with grey platelet syndrome or a patient with Glanzmann thrombasthenia. LYP7 (IgG1, F(ab')2 or Fab fragments) inhibited ristocetin-induced platelet aggregation of platelets in a dose-dependent fashion without affecting the binding of von Willebrand (vWf) factor. However, agglutination of formaldehyde-fixed platelets induced by ristocetin was not affected by monoclonal antibody LYP7. In addition, the binding of thrombin-activated platelets to neutrophils was inhibited by monoclonal antibody LYP7. These results strongly suggest that P-selectin, by promoting cell-cell contact, may play an active role in platelet-platelet interactions. PMID:8603015

  7. CYP-independent inhibition of platelet aggregation in rabbits by a mixed disulfide conjugate of clopidogrel.

    PubMed

    Zhang, H; Lauver, D A; Hollenberg, P F

    2014-12-01

    Dual antiplatelet therapy with clopidogrel and aspirin has been the standard of care in the United States for patients with acute coronary syndromes (ACS) and/or undergoing percutaneous coronary interventions (PCI). However, the effectiveness of clopidogrel varies significantly among different sub-populations due to inter-individual variability. In this study we examined the antiplatelet potential of a novel mixed disulfide conjugate of clopidogrel with the aim to overcome the inter-individual variability. In the metabolic studies using human liver microsomes and cDNA-expressed P450s, we confirmed that multiple P450s are involved in the bioactivation of 2-oxoclopidogrel to H4, one of the diastereomers of the pharmacologically active metabolite (AM) possessing antiplatelet activity. Results from kinetic studies demonstrated that 2C19 is the most active in converting 2-oxoclopidogrel to H4 with a catalytic efficiency of 0.027 µM⁻¹min⁻¹ in the reconstituted system. On the basis of this finding, we were able to biosynthesise the conjugate of clopidogrel with 3-nitropyridine-2-thiol, referred to as clopNPT, and examined its antiplatelet activity in male New Zealand white rabbits. After administration as intravenous bolus at 2 mg/kg, the clopNPT conjugate was rapidly converted to the AM leading to the inhibition of platelet aggregation (IPA). Analyses of the blood samples drawn at various time points showed that intravenous administration of clopNPT led to ~70% IPA within 1 hour and the IPA persisted for more than 3 hours. Since the antiplatelet activity of clopNPT does not require bioactivation by P450s, the mixed disulfide conjugate of clopidogrel has the potential to overcome the inter-individual variability in clopidogrel therapy. PMID:25230737

  8. The effects of the decaffeination of coffee samples on platelet aggregation in hyperlipidemic rats.

    PubMed

    Silvério, Alessandra dos Santos Danziger; Pereira, Rosemary Gualberto Fonseca Alvarenga; Lima, Adriene Ribeiro; Paula, Fernanda Borges de Araújo; Rodrigues, Maria Rita; Baldissera, Lineu; Duarte, Stella Maris da Silveira

    2013-09-01

    The effect of coffee on cardiovascular diseases is still controversial. It is known that the process of decaffeination may influence the chemical constitution and, therefore, the biological effects of coffee. This study thus evaluated the effects of decaffeination on the levels of total phenols and chlorogenic acids in Coffea arabica L. samples, as well as the effects of ingesting both integral and decaffeinated coffee on the lipid profile and hemostatic and hematological parameters in normal and hyperlipidemic rats. Samples of integral and decaffeinated lyophilized coffee (Coffea arabica L., planted in Brazil) were used for chemical analysis (total phenols, chlorogenic acid and caffeine contents). For the bioassays, coffee beverages were prepared with non-lyophilized samples (10% w/v) and were filtered and administered to animals by gavage (7.2 mL/kg/day) over 30 days. On the 31st day after beginning the treatment with coffee beverages, hyperlipidemia was induced to the animals by administering Triton WR-1339 (300 mg/kg body weight). On day 32, blood was taken to determine the lipid profile, platelet aggregation, prothrombin time, partially activated thromboplastin time and hemogram. The contents of both phenolic compounds and chlorogenic acid in the integral coffee beverage were significantly lower than those in the decaffeinated coffee beverage. The animals treated with Triton WR-1339 presented a mixed hyperlipidemia. Although the decaffeination process caused a relative increase in total phenols and chlorogenic acids, the coffee drinks were unable to change the lipid profile or the hemostatic and hematological parameters in the studied animals. PMID:23780748

  9. The effects of the decaffeination of coffee samples on platelet aggregation in hyperlipidemic rats.

    PubMed

    Silvério, Alessandra dos Santos Danziger; Pereira, Rosemary Gualberto Fonseca Alvarenga; Lima, Adriene Ribeiro; Paula, Fernanda Borges de Araújo; Rodrigues, Maria Rita; Baldissera, Lineu; Duarte, Stella Maris da Silveira

    2013-09-01

    The effect of coffee on cardiovascular diseases is still controversial. It is known that the process of decaffeination may influence the chemical constitution and, therefore, the biological effects of coffee. This study thus evaluated the effects of decaffeination on the levels of total phenols and chlorogenic acids in Coffea arabica L. samples, as well as the effects of ingesting both integral and decaffeinated coffee on the lipid profile and hemostatic and hematological parameters in normal and hyperlipidemic rats. Samples of integral and decaffeinated lyophilized coffee (Coffea arabica L., planted in Brazil) were used for chemical analysis (total phenols, chlorogenic acid and caffeine contents). For the bioassays, coffee beverages were prepared with non-lyophilized samples (10% w/v) and were filtered and administered to animals by gavage (7.2 mL/kg/day) over 30 days. On the 31st day after beginning the treatment with coffee beverages, hyperlipidemia was induced to the animals by administering Triton WR-1339 (300 mg/kg body weight). On day 32, blood was taken to determine the lipid profile, platelet aggregation, prothrombin time, partially activated thromboplastin time and hemogram. The contents of both phenolic compounds and chlorogenic acid in the integral coffee beverage were significantly lower than those in the decaffeinated coffee beverage. The animals treated with Triton WR-1339 presented a mixed hyperlipidemia. Although the decaffeination process caused a relative increase in total phenols and chlorogenic acids, the coffee drinks were unable to change the lipid profile or the hemostatic and hematological parameters in the studied animals.

  10. 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

  11. Ezetimibe's effect on platelet aggregation and LDL tendency to peroxidation in hypercholesterolaemia as monotherapy or in addition to simvastatin

    PubMed Central

    Hussein, Osamah; Minasian, LiLia; Itzkovich, Yaroslav; Shestatski, Karina; Solomon, Lizora; Zidan, Jamal

    2008-01-01

    AIMS To investigate the effect of lowering low-density lipoprotein-cholesterol (LDL-C) on platelet aggregation and LDL tendency to peroxidation by ezetimibe alone or with simvastatin in hypercholesterolaemia. METHODS Sixteen patients with LDL-C >3.4 mmol l−1 received ezetimibe for 3 months (Part I). Twenty-two patients on fixed simvastatin dose with LDL-C >2.6 mmol l−1 were enrolled (Part II). Part II patients continued simvastatin treatment 20 mg day−1 for 6 weeks, then received 20 mg day−1 simvastatin combined with ezetimibe 10 mg day−1 for another 6 weeks. The tendency of LDL to peroxidation measured by lag time in minutes required for initiation of LDL oxidation and by LDL oxidation at maximal point (plateau) was measured before and after ezetimibe treatment. RESULTS Part I: Ezetimibe 10 mg daily for 3 months decreased plasma LDL-C level 16% (P = 0.002), prolonged lag time to LDL oxidation from 144 ± 18 min to 195 ± 16 min (P < 0.001), decreasing maximal aggregation from 83 ± 15% to 60 ± 36% (P = 0.04). Part II: Serum level LDL-C decreased 23% (P = 0.02) and lag time in minutes to LDL oxidation was prolonged from 55.9 ± 16.5 to 82.7 ± 11.6 (P < 0.0001) using combined simvastatin–ezetimibe therapy. There were no differences in platelet aggregation. CONCLUSIONS Ezetimibe was associated with decreased platelet aggregation and LDL tendency to peroxidation. Treatment with ezetimibe in addition to simvastatin has an additive antioxidative effect on LDL. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Statins demonstrate a pleiotropic effect which contributes beyond the hypocholesterolaemic effect to prevent atherosclerosis. WHAT THIS STUDY ADDS Ezetimibe has an antioxidative effect when given as monotherapy or as an add-on to the statin, simvastatin. PMID:18241285

  12. Metabolomic investigation of the anti-platelet aggregation activity of ginsenoside Rk₁ reveals attenuated 12-HETE production.

    PubMed

    Ju, Hyun Kyoung; Lee, Jin Gyun; Park, Mi Kyung; Park, So-Jung; Lee, Chang Hoon; Park, Jeong Hill; Kwon, Sung Won

    2012-10-01

    Comprehensive metabolomics analysis is an effective method of measuring metabolite levels in the body following administration of a pharmaceutical compound and can allow for monitoring of the effects of the compound or assessment of appropriate treatment options for individual patients. In the present metabolomics study, samples pretreated with antiplatelet compounds were extracted and subjected to ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry. The acquired data were processed using peak clustering and evaluated by partial least-squares (PLS) and orthogonal projections to latent structures discriminant analyses (OPLS-DA). As a result, meaningful endogenous metabolites, namely eicosanoids and thromboxane B(2) (TXB(2)), were identified. TXB(2), a key element in platelet aggregation, was decreased upon ginsenoside Rk(1) treatment via inhibition of cyclooxygenase (COX) activity. One of the arachidonic acid (AA) metabolites, 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), was decreased significantly in the ginsenoside Rk(1)-treated platelets compared to the AA-induced group. In the mechanism study of ginsenoside Rk(1), a strong linkage to intracellular calcium levels, which induce platelet activation, was found. Additionally, the translocation of 12-LOX from cytosol to membrane, which is related with the intracellular calcium levels, was determined. Therefore, a decreased 12-HETE level induced by ginsenoside Rk(1) on antiplatelet aggregation is related to 12-LOX translocation resulting from decreased Ca(2+) levels. This study shows that global metabolomic analysis has potential for use in understanding the biological behavior of antiplatelet drugs. PMID:22873173

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

    PubMed

    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

    2016-06-07

    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 (r(2) = 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.

  14. 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

  15. Onion (Allium cepa L.) peel extract has anti-platelet effects in rat platelets.

    PubMed

    Ro, Ju-Ye; Ryu, Jin-Hyeob; Park, Hwa-Jin; Cho, Hyun-Jeong

    2015-01-01

    The effects of onion peel extract (OPE) in collagen (5 μg/mL)-stimulated washed rat platelet aggregation were investigated. OPE inhibited platelet aggregation via inhibition of aggregation-inducing molecules, intracellular Ca(2+) and thromboxane A2 (TXA2) by blocking cyclooxygenase-1 (COX-1) and TXA2 synthase (TXAS) activities in a dose-dependent manner. In addition, OPE elevated the formation of cyclic adenosine monophosphate (cAMP), aggregation-inhibiting molecule, but not cyclic guanosine monophosphate (cGMP). High performance liquid chromatography (HPLC) analysis of OPE revealed that OPE contains quercetin, one of the major flavonoids, which has anti-platelet effect. In conclusion, we suggest that OPE is an effective inhibitor of collagen-stimulated platelet aggregation in vitro. Therefore, it can be a promising and safe strategy for anti-cardiovascular diseases. PMID:25628983

  16. 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.

  17. 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. PMID:22797934

  18. The inhibitory mechanism of crude saponin fraction from Korean Red Ginseng in collagen-induced platelet aggregation

    PubMed Central

    Jeon, Bo Ra; Kim, Su Jung; Hong, Seung Bok; Park, Hwa-Jin; Cho, Jae Youl; Rhee, Man Hee

    2015-01-01

    Background Korean Red Ginseng has been used as a traditional oriental medicine to treat illness and to promote health for several thousand years in Eastern Asia. It is widely accepted that ginseng saponins, ginsenosides, are the major active ingredients responsible for Korean Red Ginseng’s therapeutic activity against many kinds of illness. Although the crude saponin fraction (CSF) displayed antiplatelet activity, the molecular mechanism of its action remains to be elucidated. Methods The platelet aggregation was induced by collagen, the ligand of integrin αIIβI and glycoprotein VI. The crude saponin’s effects on granule secretion [e.g., calcium ion mobilization and adenosine triphosphate (ATP) release] were determined. The activation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNKs), and p38 MAPK, and phosphoinositide 3-kinase (PI3K)/Akt was analyzed by immunoblotting. In addition, the activation of integrin αIIbβIII was examined by fluorocytometry. Results CSF strongly inhibited collagen-induced platelet aggregation and ATP release in a concentration-dependent manner. It also markedly suppressed [Ca2+]i mobilization in collagen-stimulated platelets. Immunoblotting assay revealed that CSF significantly suppressed ERK1/2, p38, JNK, PI3K, Akt, and mitogen-activated protein kinase kinase 1/2 phosphorylation. In addition, our fraction strongly inhibited the fibrinogen binding to integrin αIIbβ3. Conclusion Our present data suggest that CSF may have a strong antiplatelet property and it can be considered as a candidate with therapeutic potential for the treatment of cardiovascular disorders involving abnormal platelet function. PMID:26199561

  19. Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred

    PubMed Central

    Prevost, Nicolas; Woulfe, Donna; Tanaka, Takako; Brass, Lawrence F.

    2002-01-01

    Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and α-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation. PMID:12084815

  20. Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

    PubMed

    Metzig, C; Grabowska, E; Eckert, K; Rehse, K; Maurer, H R

    1999-01-01

    The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

  1. Testing synthetic amyloid-β aggregation inhibitor using single molecule atomic force spectroscopy.

    PubMed

    Hane, Francis T; Lee, Brenda Y; Petoyan, Anahit; Rauk, Arvi; Leonenko, Zoya

    2014-04-15

    Alzheimer's disease is a neurodegenerative disease with no known cure and few effective treatment options. The principal neurotoxic agent is an oligomeric form of the amyloid-β peptide and one of the treatment options currently being studied is the inhibition of amyloid aggregation. In this work, we test a novel pseudopeptidic aggregation inhibitor designated as SG1. SG1 has been designed to bind at the amyloid-β self-recognition site and prevent amyloid-β from misfolding into β sheet. We used atomic force spectroscopy, a nanoscale measurement technique, to quantify the binding forces between two single amyloid peptide molecules. For the first time, we demonstrate that single molecule atomic force spectroscopy can be used to assess the effectiveness of amyloid aggregation inhibitors by measuring the experimental yield of binding and can potentially be used as a screening technique for quick testing of efficacy of inhibitor drugs for amyloid aggregation.

  2. [Measurement of the concentration of free cytoplasmic calcium in the process of platelet aggregation using a fluorescent method].

    PubMed

    Popov, E G; Gavrilov, I Iu; Pozin, E Ia; Gabbasov, Z A

    1989-01-01

    A multiwavelength method for measuring free cytosolic calcium concentration is proposed. It is based on the registration of the fluorescent spectrum of calcium--sensitive probe indo-1 and deconvolution of the spectrum into components corresponding to free and bound forms of the probe. Calcium concentration is calculated as a product of calcium-probe dissociation constant by calcium-bound to free form concentration ratio. The obtained values are independent of variations in light-scattering properties of the medium and total dye concentration in the optical channel. It is shown that during ADP-induced platelet aggregation calcium concentration rises without measurable delay after the addition of the inducer and significantly decreases by the time the aggregation begins.

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

    PubMed

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

    2011-01-01

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

  4. Evaluating the inhibitory potential of Withania somnifera on platelet aggregation and inflammation enzymes: An in vitro and in silico study.

    PubMed

    M, Madhusudan; Zameer, Farhan; Naidu, Akhilender; M N, Nagendra Prasad; Dhananjaya, Bhadrapura Lakkappa; Hegdekatte, Raghavendra

    2016-09-01

    Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases. PMID:26704448

  5. Evaluating the inhibitory potential of Withania somnifera on platelet aggregation and inflammation enzymes: An in vitro and in silico study.

    PubMed

    M, Madhusudan; Zameer, Farhan; Naidu, Akhilender; M N, Nagendra Prasad; Dhananjaya, Bhadrapura Lakkappa; Hegdekatte, Raghavendra

    2016-09-01

    Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases.

  6. 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.

  7. [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.

  8. Effect of SJAMP on ATP release of platelet.

    PubMed

    Guo, T; Shen, D; Song, S; Wei, W

    1999-01-01

    The aggregation and ATP release of placelet of normal subjects were measured by platelet lumi-aggregometer. It was found that the aggregation curve induced by SJAMP at the concentration of 100 mg/L was a typical second phase aggregation. There existed a certain lag between platelet aggregation and secretion. The secretion actually began slightly after the second phase of aggregation, suggesting that the second phase aggregation induced by SJAMP is not dependent upon the release of contents of dense granule alone. If platelets were incubated with cyclo-oxygenase inhibitor, the second phase aggregation was inhibited and no ATP was released. The results indicated that the aggregation and release reaction induced by SJAMP were dependent upon the generation of prostaglandin endoperoxides and TXA2 in normal subjects. The amount of ATP release was 0.69 +/- 0.22 nmol/10(8) platelets as stimulated with SJAMP (100 mg/L). But the amount of ATP release were 1.60 +/- 0.25 and 1.37 +/- 0.15 nmol/10(8) platelets when platelets were stimulated with ADP (5 mumol/L) and collagen (5 mg/L). The amount of ATP release induced by SJAMP was significantly lower than that of ADP and collagen. These findings indicated that SJAMP was a weaker agonist than ADP in terms of platelets release reaction.

  9. Potassium 2-(1-hydroxypentyl)-benzoate inhibits ADP-induced rat platelet aggregation through P2Y1-PLC signaling pathways.

    PubMed

    Yang, Hongyan; Xu, Shaofeng; Li, Jiang; Wang, Ling; Wang, Xiaoliang

    2015-09-01

    Potassium 2-(1-hydroxypenty1)-benzoate (dl-PHPB) is a new drug candidate for treatment of ischemic stroke with antiplatelet effect. In this study, we investigated the mechanisms of dl-PHPB in inhibiting platelet aggregation. The ADP-activated P2Y1-Gq-PLC and P2Y12-Gi-AC pathways were observed, respectively. Intravenous injection of dl-PHPB (1.3, 3.9, 12.9 mg/kg) significantly inhibited ADP-, collagen-, and arachidonic acid-induced rat platelet aggregation in a dose-dependent manner, and dl-PHPB had a relatively more potent inhibitory effect on ADP-induced rat platelet aggregation than other agonists. Dl-PHPB also showed a decreased expression of CD62P (a marker for platelet activation) mediated by ADP. Both dl-PHPB and ticlopidine (P2Y12 receptor antagonist) decreased cytoplasmic Ca(2+) concentration. But, dl-PHPB did not reverse the inhibition of PGE1-induced platelet cAMP formation by ADP, which was different from ticlopidine. Further, dl-PHPB instead of ticlopidine showed increasing phospholipase C-β phosphorylation (ser(1105)). The m-3M3FBS, a phospholipase C activator, attenuated the inhibitory effect of dl-PHPB on ADP-induced platelet aggregation and enhanced IP1 accumulation in rat platelets. Dl-PHPB decreased IP1 accumulation induced by ADP but had no effect on IP1 level enhanced by m-3M3FBS. Our results suggest that dl-PHPB has a potent antiplatelet effect, which is mainly through blockade of P2Y1 receptor-PLC-IP3 pathway and decreasing cytoplasmic calcium.

  10. 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

  11. Discovery of Potent and Selective Inhibitors of Human Platelet type 12-Lipoxygenase

    PubMed Central

    Kenyon, Victor; Rai, Ganesha; Jadhav, Ajit; Schultz, Lena; Armstrong, Michelle; Jameson, J. Brian; Perry, Steven; Joshi, Netra; Bougie, James M.; Leister, William; Taylor-Fishwick, David A.; Nadler, Jerry L.; Holinstat, Michael; Simeonov, Anton; Maloney, David J.; Holman, Theodore R.

    2011-01-01

    We report the discovery of novel small molecule inhibitors of platelet type 12-human lipoxygenase, which display nanomolar activity against the purified enzyme, using a quantitative high throughput screen (qHTS) on a library of 153,607 compounds. These compounds also exhibit excellent specificity, >50-fold selectivity vs. the paralogs, 5-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity vs. ovine cyclooxygenase-1 and human cyclooxygenase-2. Kinetic experiments indicate this chemotype is a non-competitive inhibitor that does not reduce the active site iron. Moreover, chiral HPLC separation of two of the racemic lead molecules revealed a strong preference for the (–)-enantiomers (IC50 of 0.43 +/- 0.04 and 0.38 +/- 0.05 μM) compared to the (+)-enantiomers (IC50 of >25 μM for both), indicating a fine degree of selectivity in the active site due to chiral geometry. In addition, these compounds demonstrate efficacy in cellular models, which underscores their relevance to disease modification. PMID:21739938

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25759392

  13. Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans

    PubMed Central

    Carneiro, Ana Marin D.; Cook, Edwin H.; Murphy, Dennis L.; Blakely, Randy D.

    2008-01-01

    The essential contribution of the antidepressant-sensitive serotonin (5-HT) transporter SERT (which is encoded by the SLC6A4 gene) to platelet 5-HT stores suggests an important role of this transporter in platelet function. Here, using SERT-deficient mice, we have established a role for constitutive SERT expression in efficient ADP- and thrombin-triggered platelet aggregation. Additionally, using pharmacological blockers of SERT and the vesicular monoamine transporter (VMAT), we have identified a role for ongoing 5-HT release and SERT activity in efficient human platelet aggregation. We have also demonstrated that fibrinogen, an activator of integrin αIIbβ3, enhances SERT activity in human platelets and that integrin αIIbβ3 interacts directly with the C terminus of SERT. Consistent with these findings, knockout mice lacking integrin β3 displayed diminished platelet SERT activity. Conversely, HEK293 cells engineered to express human SERT and an activated form of integrin β3 exhibited enhanced SERT function that coincided with elevated SERT surface expression. Our results support an unsuspected role of αIIbβ3/SERT associations as well as αIIbβ3 activation in control of SERT activity in vivo that may have broad implications for hyperserotonemia, cardiovascular disorders, and autism. PMID:18317590

  14. Maintaining Moderate Platelet Aggregation and Improving Metabolism of Endothelial Progenitor Cells Increase the Patency Rate of Tissue-Engineered Blood Vessels.

    PubMed

    Wu, Yangxiao; Li, Li; Chen, Wen; Zeng, Wen; Zeng, Lingqin; Wen, Can; Zhu, Chuhong

    2015-07-01

    Small-diameter tissue-engineered blood vessels (TEBVs) have been associated with low, long-term patency rates primarily because of acute thrombosis in early stages and an inability to achieve early endothelialization. Platelets and endothelial progenitor cells (EPCs) play a key role in these processes. A nano delayed-release 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR)-bound TEBV was implanted in rat carotid arteries for 3 months. AICAR-bound TEBVs had a high patency rate compared with control TEBVs after 3 months. We found that AICAR maintained moderate platelet aggregation in vivo. In vitro data indicated that AICAR inhibits the release of 5-hydroxytryptamine and thromboxane A2 in activating platelets to reduce platelet aggregation. Then, we confirmed that AICAR strengthens the EPC energy state, which results in earlier endothelialization. The homing, migration, and paracrine function of EPCs were enhanced by AICAR in vitro. Besides, AICAR can contribute to the migration of endothelial cells near the anastomosis. The cellularization of TEBVs at different time points was observed too. In conclusion, our study suggests that the application of nanodelivery material containing AICAR can effectively improve small-diameter TEBVs by maintaining moderate platelet aggregation and improving metabolism of EPCs.

  15. 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

  16. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:26343633

  18. Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase.

    PubMed

    Kim, Soochong; Kunapuli, Satya P

    2011-07-01

    Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways. PMID:21592972

  19. The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection.

    PubMed

    Nkambule, Bongani B; Davison, Glenda; Ipp, Hayley

    2015-01-01

    Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96-29.36] vs. control 2.48[1.56-6.04]; p < 0.0001). In addition, the HIV group showed higher CD62P MFI levels (HIV CD62P MFI 3.25 ± 7.23 vs. control 2.35 ± 1.31, p = 0.0292). Baseline levels of %CD36 expression were significantly higher in HIV-positive patients (%CD36 12.41[6.31-21.83] vs. control 6.04[1.34-13.15]; p = 0.0091). However, the baseline CD36MFI showed no significant difference between the two groups (HIV CD36 MFI 3.09 ± 0.64 vs. control 2.44 ± 0.11, p = 0.4591). The HIV group showed higher levels of % CD36

  20. A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor.

    PubMed

    Pollitt, Sonia K; Pallos, Judit; Shao, Jieya; Desai, Urvee A; Ma, Aye Aye K; Thompson, Leslie Michels; Marsh, J Lawrence; Diamond, Marc I

    2003-11-13

    Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing. PMID:14622574

  1. 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.

  2. 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. PMID:27548633

  3. Effect of allicin (diallyl disulfide-oxide) on prostaglandin endoperoxide H/sub 2/ (PGH/sub 2/) and arachidonic acid (AA) metabolism and platelet aggregation

    SciTech Connect

    Mayeux, P.R.; Agrawal, K.C.; King, B.T.; Kadowitz, P.J.; McNamara, D.B.

    1986-03-01

    The authors report here the effects of pure allicin (the antibacterial component of GO), synthesized from diallyl disulfide and hydrogen peroxide, on human platelet aggregation, PGH/sub 2/ metabolism in microsomes of bovine lung (BL) and bovine coronary artery (BCA), homogenates of human platelet (HP), and on AA metabolism in HP. Allicin at 16 ..mu..g/ml to 160 ..mu..g/ml produced concentration-dependent inhibition of platelet aggregation to 1.6 mM AA and 2.8 ..mu..M U 46619, a stable analog of PGH/sub 2/ and a TXA/sub 2/ minic. BL (200 ..mu..g protein), BCA (10 ..mu..g protein), and HP (1500 ..mu..g protein) were incubated with 10 ..mu..M (/sup 14/C) PGH/sub 2/ +/- allicin. HP (1500 ..mu..g protein) were incubated with 20 ..mu..M (/sup 14/C) AA +/- allicin. Products were separated by TLC and quantified by radiochromatographic scan. Allicin in the concentration range of 10-/sup 6/M-10-/sup 3/M induced no change in the formation of prostacyclin by BL and BCA or of TXA/sub 2/ by BL and HP. These data suggest that the platelet antiaggregatory action of allicin is not due to inhibition of cyclooxygenase or TXA/sub 2/ synthetase in the human platelet, but may be related to interactions at the TXA/sub 2/ receptor or on cyclic nucleotide levels.

  4. Selective anti-platelet aggregation synergism between a prostacyclin-mimetic, RS93427 and the nitrodilators sodium nitroprusside and glyceryl trinitrate.

    PubMed

    Willis, A L; Smith, D L; Loveday, M; Fulks, J; Lee, C H; Hedley, L; VanAntwerp, D

    1989-12-01

    1. Citrated platelet-rich plasma from human donors was used to examine turbidometrically the platelet aggregation response to collagen (2.5 micrograms ml-1) and ADP (1.6 microgram ml-1). 2. With collagen as an aggregating agent, the limited (35% maximal inhibition) inhibitory effects of glyceryl trinitrate (GTN, 0.78-50 micrograms ml-1) were markedly potentiated by threshold (3.3-10 ng ml-1) concentrations of RS93427, an orally active prostacyclin-mimetic. Almost complete inhibition of aggregation could then be produced. 3. A threshold concentration of RS93427 (3.3 ng ml-1) similarly potentiated the ability of sodium nitroprusside (NaNp, 0.78-10 micrograms ml-1) to inhibit collagen-induced platelet aggregation. There was an 8 fold reduction in the IC25 concentration of NaNp. 4. Threshold concentrations of the nitrodilators were also able to potentiate the anti-aggregatory effects of RS93427 (0.03-30 ng ml-1) on collagen-induced platelet aggregation. With threshold concentrations of either GTN (6.3-25 micrograms ml-1) or NaNp (0.3-1.3 microgram ml-1), the mean IC50 concentration of RS93427 was reduced 4 or 6 fold, respectively, while the IC25 concentration was reduced 6 or 10 fold, respectively. 5. No similar synergistic interactions were seen between RS93427 and the nitrodilators when ADP was used as an aggregating agent. 6. In spontaneously hypertensive rats, the dose-response for the hypotensive response to bolus doses of RS93427 was not altered by concomitant steady state infusion of a threshold dose (1 micrograms kg-1 min-1) of GTN. 7. Possible therapeutic implications of these findings are discussed.

  5. Targeting the anionic region of human protease activated receptor 4 (PAR4) inhibits platelet aggregation and thrombosis without interfering with hemostasis

    PubMed Central

    Mumaw, M. M.; de la Fuente, M.; Noble, D. N.; Nieman, M.T.

    2014-01-01

    Summary Background Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations including bleeding risk. Objective The aim of the current study is to evaluate the PAR4 anionic cluster as a viable antiplatelet target using a polyclonal antibody (CAN12). Methods We used western blotting, aggregation, and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis. Results We show that CAN12 is able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibits thrombin induced human platelet aggregation and secretion in a dose dependent manner. We next determined that the specificity of CAN12 is agonist dependent. In vivo, we determined that CAN12 is able to inhibit arterial thrombosis and using two independent methods, we found that CAN12 does not influence hemostasis. Conclusion Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an anti-platelet therapy. PMID:24888424

  6. 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.

  7. 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.

  8. In-vitro model for the ultrastructural study of the formation of thrombi in human platelets.

    PubMed

    Cerecedo, Doris; González, Sirenia; Mondragón, Mónica; Reyes, Elba; Mondragón, Ricardo

    2006-03-01

    Platelets are cell fragments with dynamic properties involved in clot formation after tissue damage. Platelet activation causes a change in shape, secretion of intracellular granules and aggregation with each other through the cytoskeleton components and biochemical changes. Platelet adhesion, considered as the major event in haemostasis, has been studied in several in-vitro and in-vivo models to evaluate the feasible thrombogenicity of some materials, the dynamics of specific receptors, as well as the effect of different buffers and inhibitors in this process. In spite of the numerous reports about platelet activation, to date there is no information available about the fine structure of the platelet-platelet and platelet-substrate interactions. In the present report we describe an in-vitro system that allows the visualization of these interactions: platelets are adhered to an inert substrate, and interactions with suspended platelets as a process to initiate the formation of thrombi was followed by ultramicrotomy and transmission electron microscopy.

  9. 2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a direct thrombin inhibitor with a suppressive effect on thromboxane formation in platelets.

    PubMed

    Wu, Chin-Chung; Wang, Tsai-Wei; Wang, Wei-Ya; Hsieh, Pei-Wen; Wu, Yang-Chang

    2005-12-19

    2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a newly synthetic benzoxazinone derivative, has previously been shown to inhibit rabbit platelet aggregation caused by thrombin and arachidonic acid. In the present study, the mechanism for the antiplatelet effect of HPW-RX2 was further investigated. In human platelets, HPW-RX2 concentration-dependently inhibited platelet aggregation, ATP release, P-selectin expression, and intracellular calcium mobilization caused by thrombin. In contrast, HPW-RX2 had no significant effect on either SFLLRN- or GYPGKF-induced platelet aggregation, indicating that HPW-RX2 did not interfere with platelet thrombin receptors. Moreover, HPW-RX2 inhibited the amidolytic activity of thrombin and prolonged the fibrinogen clotting time. These results suggest that the inhibitory effect of HPW-RX2 on thrombin-induced platelet aggregation is via direct inhibition of thrombin proteolytic activity. Besides the inhibition on thrombin, HPW-RX2 also prevented platelet aggregation, ATP release, and increase in [Ca2+]i caused by arachidonic acid and low concentration collagen. In a parallel manner, both arachidonic acid-induced thromboxane B2 and prostaglandin D2 formations were decreased in platelets treated with HPW-RX2. This indicates that HPW-RX2 is able to inhibit the arachidonic acid cascade at the cyclooxygenase level. This is the first report of a benzoxazinone derivative possessing both thrombin and cyclooxygenase inhibitory properties. The dual effect of HPW-RX2 might provide extra therapeutic benefits for treatment of arterial thrombosis. PMID:16313903

  10. Inhibition of whole blood platelet-aggregation by compounds in garlic clove extracts and commercial garlic products.

    PubMed

    Lawson, L D; Ransom, D K; Hughes, B G

    1992-01-15

    The inhibitory effects of adenosine and 16 quantitatively determined organosulfur compounds derived from garlic cloves or commercial garlic preparations on collagen stimulated in vitro platelet aggregation in whole blood were determined. An estimation of the anti-aggregatory activity of several brands of the major types of commercial garlic preparations was determined from the activities of the individual compounds present in each sample. In platelet rich plasma (PRP) most of the anti-aggregatory activity of garlic clove homogenates was due to adenosine; however, in whole blood neither adenosine nor the polar fraction had any effect and all of the anti-aggregatory activity was due to allicin and other thiosulfinates. Allicin was equally active in whole blood and PRP. Among brands there was a several-fold variation in content of the organosulfur compounds and activity for all types of garlic products tested. The best garlic powder tablets were equally as active as clove homogenates whereas steam-distilled oils were 35% as active and oil-macerates (due to low content) only 12% as active. A garlic product aged many months in aqueous alcohol had no activity. For steam-distilled oils, most of the activity was due to diallyl trisulfide. For the oil-macerates, most of the activity was due largely to the vinyl dithiins. Ajoene, an exclusive component of the oil-macerates, had highest specific activity of all the compounds tested but, because of its low concentration, had only 13% of the activity of diallyl trisulfide and 3% of the activity of allicin. Compounds which may be active in vivo are discussed. PMID:1579891

  11. [Prophylactic platelet transfusions].

    PubMed

    Ilmakunnas, Minna; Remes, Kari; Hiippala, Seppo; Mäkisalo, Heikki; Åberg, Fredrik

    2016-01-01

    The consumption of platelet products in Finland is exceptionally high. For the most part, platelets are transfused pre-operatively to thrombocytopenic patients in order to prevent hemorrhage. Most of the minor procedures could, however, be conducted even if the patients'platelet levels would be lower than usual. In cardiac surgery, platelets are used because of the hemorrhagic diathesis associated with platelet inhibitors. Platelet inhibitors will, however, also bind to transfused platelets, whereby instead of prophylactic platelet transfusions it would be more sensible to leave the thorax open and not carry out ineffective platelet transfusions until the effect of the inhibitors has run out. We outline the prophylactic use of platelets based on recent international clinical practice guidelines. PMID:27400590

  12. 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

  13. Development of dual targeting inhibitors against aggregations of amyloid-β and tau protein.

    PubMed

    Fuse, Shinichiro; Matsumura, Keisuke; Fujita, Yuki; Sugimoto, Hachiro; Takahashi, Takashi

    2014-10-01

    Aggregations of both amyloid-β (Aβ) and hyper-phosphorylated tau proteins are recognized as key pathological manifestations of Alzheimer's disease (AD). Agents that inhibit both those forms of aggregation show promise as drug candidates. Seventeen oligo heteroaromatic compounds were rapidly synthesized via a one-pot, 3- or 4-component coupling procedure. Evaluations showed that compounds E16 and E18 were the most potent inhibitors of Aβ and tau aggregations (E16: IC50s = 0.38, 0.29 μM against Aβ, tau, respectively, E18: IC50s = 0.55, 0.30 μM against Aβ, tau, respectively).

  14. Development of a new distyrylbenzene-derivative amyloid-β-aggregation and fibril formation inhibitor.

    PubMed

    Suzuki, Hideharu; Ishigami, Akihito; Orimoto, Ayako; Matsuyama, Akihiro; Handa, Setsuko; Maruyama, Naoki; Yokoyama, Yuusaku; Okuno, Hiroaki; Nakakoshi, Masamichi

    2012-01-01

    Several new amyloid-β (Aβ) aggregation inhibitors were synthesized according to our theory that a hydrophilic moiety could be attached to the Aβ-recognition unit for the purpose of preventing amyloid plaque formation. A distyrylbenzene-derivative, DSB(EEX)(3), which consider the Aβ recognition unit (DSB, 1,4-distyrylbenzene) and expected to bind to amyloid fibrils (β-sheet structure), was combined with the hydrophilic aggregation disrupting element (EEX) (E, Glu; X, 2-(2-(2-aminoethoxy)ethoxy)acetic acid). This DSB(EEX)(3) compound, compared to several others synthesized similarly, was found to be the most active for reducing Aβ toxicity toward IMR-32 human neuroblastoma cells. Moreover, its inhibition of Aβ-aggregation or fibril formation was directly confirmed by transmission electron microscopy and atomic force microscopy. These results suggest that the Aβ aggregation inhibitor DSB(EEX)(3) disrupts clumps of Aβ protein and is a likely candidate for drug development to treat Alzheimer's disease.

  15. Platelet Inhibitors in Non-ST-Segment Elevation Acute Coronary Syndromes and Percutaneous Coronary Intervention: Glycoprotein IIb/IIIa Inhibitors, Clopidogrel, or Both?

    PubMed Central

    Silva, Matthew A; Donovan, Jennifer L; Gandhi, Pritesh J; Volturo, Gregory A

    2006-01-01

    The role of glycoprotein (Gp) IIb/IIIa receptor antagonists remains controversial and these agents are infrequently utilized during non-ST-segment elevation acute coronary syndromes (NSTE-ACS) despite American Heart Association/American College of Cardiology guidelines. Despite recommendations, the NRMI-4 (National Registry of Myocardial Infarction 4) and CRUSADE (Can rapid risk stratification of unstable angina patients suppress adverse outcomes with early implementation of the ACC/AHA guidelines?) registries observed that only 25%–32% of eligible patients received early Gp IIb/IIIa therapy, despite a 6.3% absolute mortality reduction in NRMI-4 and a 2% absolute mortality reduction in CRUSADE. A pooled analysis of Gp IIb/IIIa data from these registries suggest a major reduction in mortality (Odds Ratio = 0.43, 95% Confidence Index 0.25–0.74, p = 0.002) with early Gp IIb/IIIa therapy, yet clinicians fail to utilize this option in NSTE-ACS. The evidence-based approach to NSTE-ACS involves aspirin, clopidogrel, low-molecular weight heparins, or unfractionated heparin in concert with Gp IIb/IIIa receptor antagonists, however, newer percutaneous coronary intervention (PCI)-based trials challenge current recommendations. Novel strategies emerging in NSTE-ACS include omitting Gp IIb/IIIa inhibitors altogether or using Gp IIb/IIIa inhibitors with higher doses of clopidogrel in selected patients. The ISAR-REACT (Intracoronary stenting and antithrombotic regimen–Rapid early action for coronary treatment) and ISAR-SWEET (ISAR–Is abciximab a superior way to eliminate elevated thrombotic risk in diabetics) trials question the value of abciximab when 600 mg of clopidogrel concurrently administered during PCI. The CLEAR-PLATELETS (Clopidogrel loading with eptifibatide to arrest the reactivity of platelets) and PEACE (Platelet activity extinction in non-Q-wave MI with ASA, clopidogrel, and eptifibatide) trials suggest more durable platelet inhibition when Gp IIb

  16. Albocollagenase, a novel recombinant P-III snake venom metalloproteinase from green pit viper (Cryptelytrops albolabris), digests collagen and inhibits platelet aggregation.

    PubMed

    Pinyachat, Anuwat; Rojnuckarin, Ponlapat; Muanpasitporn, Chuanchom; Singhamatr, Pon; Nuchprayoon, Surang

    2011-04-01

    Molecular cloning and functional characterization of P-III snake venom metalloproteinases (SVMPs) will give us deeper insights in the pathogenesis of viper bites. This may lead to novel therapy for venom-induced local tissue damages, the complication refractory to current antivenom. The aim of this study was to elucidate the in vitro activities of a new SVMP from the green pit viper (GPV) using recombinant DNA technology. We report, here, a new cDNA clone from GPV (Cryptelytrops albolabris) venom glands encoding 614 amino acid residues P-III SVMP, termed albocollagenase. The conceptually translated protein comprised a signal peptide and prodomain, followed by a metalloproteinase domain containing a zinc-binding motifs, HEXGHXXGXXH-CIM and 9 cysteine residues. The disintegrin-like and cysteine-rich domains possessed 24 cysteines and a DCD (Asp-Cys-Asp) motif. The albocollagenase deduced amino acid sequence alignments showed approximately 70% identity with other P-III SVMPs. Notably, the prodomain was highly conserved, while the metalloproteinase, disintegrin-like and cysteine-rich domains contained several differences. Albocollagenase without the signal peptide and prodomain was expressed in Pichia pastoris with an N-terminal six-histidine tag. After affinity purification from the supernatant of methanol-induced media, SDS-PAGE and Western blot analysis in both reducing and non-reducing conditions showed a protein band of approximately 62 kDa. The recombinant albocollagenase could digest human type IV collagen from human placenta basement membrane within 1 min. After 10-min incubation, it also inhibited collagen-induced platelet aggregation with 50% inhibitory concentration (IC₅₀) of 70 nM. This is the first report of the active recombinant SVMP enzymes expressed in P. pastoris. The results suggest the significant roles of P-III SVMP in local and systemic pathology of envenomated patients. Inhibitors of this SVMP will be investigated in further studies to find a

  17. Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom.

    PubMed Central

    Leduc, M; Bon, C

    1998-01-01

    Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, alpha and beta, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the alpha chain and 125 amino acids for the beta chain. These polypeptides include a carbohydrate-recognition domain (CRD) in which some of the specific amino acids required for binding Ca2+ and galactose or mannose are absent. The presence of such a domain means that CVX can be included in the family of C-type lectins along with other snake venom proteins, although it is not a true lectin. Assuming that the localization of intracatenary disulphide bridges of each CVX chain is similar to that of the CRD and that an intercatenary bridge between the alpha and beta chains is similar to that of the C-type lectin botrocetin, we postulate the existence of an additional intercatenary bridge, which explains the tridimeric structure (alphabeta)3 of CVX. PMID:9657980

  18. Effects of combination treatment with policosanol and omega-3 fatty acids on platelet aggregation: A randomized, double-blind clinical study

    PubMed Central

    Castaño, Gladys; Arruzazabala, Maria L.; Fernández, Lilia; Mas, Rosa; Carbajal, Daisy; Molina, Vivian; Illnait, José; Mendoza, Sarahí; Gámez, Rafael; Mesa, Melbis; Fernández, Julio

    2006-01-01

    Background: Policosanol is a mixture of long-chain primary aliphatic alcoholspurified from sugar cane wax that has cholesterol lowering and antiplatelet effects. Omega-3 fatty acids (FA) have triglyceride lowering and antiplatelet effects. Combination treatment with policosanol and omega-3 FA (Ω23FA) has been associated with significant inhibition of platelet aggregation in rabbits compared with either drug alone. Objective: The aim of this study was to investigate the effects of combination treatment with Ω3FA (1 g/d) and policosanol (Ω3FA+Poli) compared with Ω3FA (1 g/d) plus placebo (Ω3FA+Pla) on platelet aggregation in human patients with hypercholesterolemia. Methods: This randomized, double-blind, clinical study at the Surgical Medical Research Center (Havana City, Cuba) recruited outpatients from lipid clinics, with some atherosclerotic risk factors. Outpatients of both sexes aged 20 to 75 years with serum total cholesterol (TC) levels ≥5 and <6 mmol/L were eligible to enroll. They were included in the study at the end of a 4-week diet stabilization period if their platelet aggregation to arachidonic acid (AA) was ≥50% and serum TC level remained ≥5 mmol/L. Patients were then evenly randomized to receive Ω3FA (1 g/d) + placebo or Ω3FA (1 g/d) + policosanol (10 mg/d) to be taken PO with the evening meal for 21 days. Treatment was assigned according to a randomization code using balanced blocks and a 1:1 allocation ratio. Inhibition of platelet aggregation to AA was the primary efficacy variable, while effects on platelet aggregation to collagen and epinephrine and on lipid profile were secondary variables. Drug compliance and adverse events (AEs) were monitored. Tolerability was assessed using physical examinations and laboratory test results. Results: Sixty-four subjects were initially enrolled. Fifty-four patients (30 women, 24 men; mean [SD] age, 58.4 [12] years, [range, 40–70 years]) met the inclusion criteria and were randomized to

  19. Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation

    PubMed Central

    Yeung, Jennifer; Apopa, Patrick L.; Vesci, Joanne; Kenyon, Victor; Rai, Ganesha; Jadhav, Ajit; Simeonov, Anton; Holman, Theodore R.; Maloney, David J.; Boutaud, Olivier

    2012-01-01

    Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis, which is a major cause of myocardial infarction and stroke. After activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation because inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity. Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet. To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured, including platelet aggregation, calcium mobilization, and integrin activation. Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ3 activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Furthermore, inhibition of 12-LOX had no effect on PKC-mediated aggregation, indicating that 12-LOX is upstream of PKC. These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for antiplatelet therapy. PMID:22155783

  20. β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor.

    PubMed

    Mirecka, Ewa A; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2016-01-01

    In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

  1. β-Hairpin of Islet Amyloid Polypeptide Bound to an Aggregation Inhibitor

    PubMed Central

    Mirecka, Ewa A.; Feuerstein, Sophie; Gremer, Lothar; Schröder, Gunnar F.; Stoldt, Matthias; Willbold, Dieter; Hoyer, Wolfgang

    2016-01-01

    In type 2 diabetes, the formation of islet amyloid consisting of islet amyloid polypeptide (IAPP) is associated with reduction in β-cell mass and contributes to the failure of islet cell transplantation. Rational design of inhibitors of IAPP amyloid formation has therapeutic potential, but is hampered by the lack of structural information on inhibitor complexes of the conformationally flexible, aggregation-prone IAPP. Here we characterize a β-hairpin conformation of IAPP in complex with the engineered binding protein β-wrapin HI18. The β-strands correspond to two amyloidogenic motifs, 12-LANFLVH-18 and 22-NFGAILS-28, which are connected by a turn established around Ser-20. Besides backbone hydrogen bonding, the IAPP:HI18 interaction surface is dominated by non-polar contacts involving hydrophobic side chains of the IAPP β-strands. Apart from monomers, HI18 binds oligomers and fibrils and inhibits IAPP aggregation and toxicity at low substoichiometric concentrations. The IAPP β-hairpin can serve as a molecular recognition motif enabling control of IAPP aggregation. PMID:27641459

  2. New mimetic peptides inhibitors of Αβ aggregation. Molecular guidance for rational drug design.

    PubMed

    Barrera Guisasola, Exequiel E; Andujar, Sebastián A; Hubin, Ellen; Broersen, Kerensa; Kraan, Ivonne M; Méndez, Luciana; Delpiccolo, Carina M L; Masman, Marcelo F; Rodríguez, Ana M; Enriz, Ricardo D

    2015-05-01

    A new series of mimetic peptides possessing a significant Aβ aggregation modulating effect was reported here. These compounds were obtained based on a molecular modelling study which allowed us to perform a structural-based virtual selection. Monitoring Aβ aggregation by thioflavin T fluorescence and transmission electron microscopy revealed that fibril formation was significantly decreased upon prolonged incubation in presence of the active compounds. Dot blot analysis suggested a decrease of soluble oligomers strongly associated with cognitive decline in Alzheimer's disease. For the molecular dynamics simulations, we used an Aβ42 pentameric model where the compounds were docked using a blind docking technique. To analyze the dynamic behaviour of the complexes, extensive molecular dynamics simulations were carried out in explicit water. We also measured parameters or descriptors that allowed us to quantify the effect of these compounds as potential inhibitors of Aβ aggregation. Thus, significant alterations in the structure of our Aβ42 protofibril model were identified. Among others we observed the destruction of the regular helical twist, the loss of a stabilizing salt bridge and the loss of a stabilizing hydrophobic interaction in the β1 region. Our results may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules and might provide a guide in the design of new aggregation modulating ligands.

  3. The effects of a selective 5-HT2 receptor antagonist (ICI 170,809) on platelet aggregation and pupillary responses in healthy volunteers.

    PubMed Central

    Millson, D S; Jessup, C L; Swaisland, A; Haworth, S; Rushton, A; Harry, J D

    1992-01-01

    1. ICI 170,809 (2-(2-dimethylamino-2-methylpropylthio)-3-phenylquinoline hydrochloride) is a potent 5-hydroxytryptamine (5-HT) type 2 postsynaptic receptor antagonist. 2. Effects of ICI 170,809 as single oral doses (3, 7, 15 and 30 mg) or placebo were studied on the duration of antagonism for the ex vivo platelet aggregatory response to 5-HT and to the pupillary light constrictor response in eight healthy male volunteers. 3. Pupillary dark adapted responses to a 0.5 s light stimulus were measured using a portable infrared pupillometer, for up to 24 h after dosing. 4. The in vitro platelet 5-HT aggregation response was reduced by ICI 170,809, with depression of the dose-response curve to 5-HT at all concentrations of 5-HT and with no evidence for a parallel shift. 5. The ex vivo platelet 5-HT response demonstrated a dose related significant (P less than 0.02) decrease in aggregation reaching a maximum at 2 h after dosing with the effect persisting for at least 8 h after dosing with the 7 and 15 mg doses. 6. Resting pupil diameter (RPD), and light induced pupillary responses in the dark adapted pupil, showed a significant (P less than 0.01) dose related reduction with significant (P less than 0.05) effects still present with the 15 and 30 mg doses at 8 h after dosing. 7. We conclude that, changes in both ex vivo platelet aggregation to 5-HT and dark adapted pupil size, are significantly correlated (P less than 0.0001) with log plasma concentrations (ng ml-1) of ICI 170,809, enabling the assessment of 5-HT2-receptor antagonism in man. PMID:1576048

  4. Reduced IL-35 levels are associated with increased platelet aggregation and activation in patients with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

    PubMed

    Zhang, Xiaohui; Zhou, Yi; Xu, Lanping; Han, Wei; Chen, Huan; Chen, Yuhong; Fu, Haixia; Zhou, Shiyuan; Zhao, Jingzhong; Wang, Qianming; Feng, Feier; Zhu, Xiaolu; Liu, Kaiyan; Huang, Xiaojun

    2015-05-01

    Acute graft-versus-host disease (aGVHD) is a major complication associated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Interleukin (IL)-35 is a novel anti-inflammatory cytokine that suppresses the immune response. This prospective study explored IL-35 plasma levels in 65 patients after HSCT. The results revealed that the peripheral blood of patients with grades III-IV aGVHD (23.46 ng/ml) had reduced IL-35 compared to transplanted patients with grades I-II aGVHD (40.26 ng/ml, p < 0.01) or patients without aGVHD (41.40 ng/ml, p < 0.05). Allografts, including granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cell (PBPC) and G-CSF-primed bone marrow (GBM), from 38 patients were analyzed for IL-35 levels with respect to aGVHD. The patients who received lower levels of IL-35 cells in the GBM (28.0 ng/ml, p = 0.551) or lower levels of IL-35 in PBPC (53.46 ng/ml, p = 0.03) exhibited a higher incidence of aGVHD. Patients with aGVHD have increased platelet aggregation. IL-35 was added to patient blood in vitro, and platelet aggregation was inhibited by IL-35 in a dose-dependent manner. The markers of platelet activation (CD62P/PAC-1) can also be inhibited by IL-35. The results indicate that IL-35 may affect the development of aGVHD by inhibiting platelet activation and aggregation. Our data suggests that IL-35 represents a potentially effective therapeutic agent against aGVHD after allo-HSCT.

  5. Pharmacological characterization of nanoparticle-induced platelet microaggregation using quartz crystal microbalance with dissipation: comparison with light aggregometry

    PubMed Central

    Santos-Martinez, Maria J; Tomaszewski, Krzysztof A; Medina, Carlos; Bazou, Despina; Gilmer, John F; Radomski, Marek W

    2015-01-01

    Background Engineered nanoparticles (NPs) can induce platelet activation and aggregation, but the mechanisms underlying these interactions are not well understood. This could be due in part to use of devices that study platelet function under quasi-static conditions with low sensitivity to measure platelet microaggregation. Therefore, in this study we investigated the pharmacological pathways and regulators of NP-induced platelet microaggregation under flow conditions at nanoscale using quartz crystal microbalance with dissipation (QCM-D) and compared the data thus obtained with those generated by light aggregometry. Methods Blood was collected from healthy volunteers, and platelet-rich plasma was obtained. Thrombin receptor-activating peptide, a potent stimulator of platelet function, and pharmacological inhibitors were used to modulate platelet microaggregation in the presence/absence of silica (10 nm and 50 nm) and polystyrene (23 nm) NPs. Light aggregometry was used to study platelet aggregation in macroscale. Optical, immunofluorescence, and scanning electron microscopy were also used to visualize platelet aggregates. Results Platelet microaggregation was enhanced by thrombin receptor-activating peptide, whereas prostacyclin, nitric oxide donors, acetylsalicylic acid, and phenanthroline, but not adenosine diphosphate (ADP) blockers, were able to inhibit platelet microaggregation. NPs caused platelet microaggregation, an effect not detectable by light aggregometry. NP-induced microaggregation was attenuated by platelet inhibitors. Conclusion NP-induced platelet microaggregation appears to involve classical proaggregatory pathways (thromboxane A2-mediated and matrix metalloproteinase-2-mediated) and can be regulated by endogenous (prostacyclin) and pharmacological (acetylsalicylic acid, phenanthroline, and nitric oxide donors) inhibitors of platelet function. Quartz crystal microbalance with dissipation, but not light aggregometry, is an appropriate method for

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

    PubMed

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

    2006-10-01

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

  7. 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

    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 (81mg/day) for seven days, then with FO (4g/day) for 28 days, then in combination for another seven days. Lysophospholipids and platelet measures were determined after acute (4h) 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Medvedev, I N; Skoriatina, I A

    2010-01-01

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

  10. Selective inhibition of the platelet phosphoinositide 3-kinase p110beta as promising new strategy for platelet protection during extracorporeal circulation.

    PubMed

    Straub, Andreas; Wendel, Hans Peter; Dietz, Klaus; Schiebold, Daniela; Peter, Karlheinz; Schoenwaelder, Simone M; Ziemer, Gerhard

    2008-03-01

    Extracorporeal circulation (ECC) is used in cardiac surgery for cardiopulmonary bypass as well as in ventricular assist devices and for extracorporeal membrane oxygenation. Blood contact with the artificial surface and shear stress of ECC activates platelets and leukocytes resulting in a coagulopathy and proinflammatory events. Blockers of the platelet glycoprotein (GP) IIb/IIIa (CD41/CD61) can protect platelet function during ECC, a phenomenon called "platelet anaesthesia", but may be involved in post-ECC bleeding. We hypothesized that the new selective phosphoinositide 3-kinase p110beta inhibitor TGX-221 that inhibits shear-induced platelet activation without prolonging the bleeding time in vivo may also protect platelet function during ECC. Heparinized blood of healthy volunteers (n = 6) was treated in vitro with either the GP IIb/IIIa blocker tirofiban, TGX-221 or as control and circulated in an ECC model. Before and after 30 minutes circulation CD41 expression on the ECC-tubing as measure for platelet-ECC binding and generation of the platelet activation marker beta-thromboglobulin were determined using ELISA. Platelet aggregation and platelet-granulocyte binding were analysed in flow cytometry. After log-transforming the data statistical evaluation was performed using multifactor ANOVA in combination with Tukey's HSD test (global alpha = 5%). Tirofiban and TGX-221 inhibited platelet-ECC interaction, platelet aggregation and platelet-granulocyte binding. Tirofiban also inhibited ECC-induced beta-thromboglobulin release. The observed inhibition of platelet-ECC interaction and platelet activation by tirofiban contributes to explain the mechanism of "platelet anaesthesia". TGX-221 represents a promising alternative to GP IIb/IIIa blockade and should be further investigated for use during ECC in vivo.

  11. Impact of Dabigatran versus Phenprocoumon on ADP Induced Platelet Aggregation in Patients with Atrial Fibrillation with or without Concomitant Clopidogrel Therapy (the Dabi-ADP-1 and Dabi-ADP-2 Trials)

    PubMed Central

    Martischnig, Amadea M.; Mehilli, Julinda; Pollak, Janina; Petzold, Tobias; Fiedler, Anette K.; Mayer, Katharina; Schulz-Schüpke, Stefanie; Sibbing, Dirk; Massberg, Steffen; Kastrati, Adnan; Sarafoff, Nikolaus

    2015-01-01

    Background. A relevant number of patients receive triple therapy with clopidogrel, aspirin, and oral anticoagulation. Clopidogrel's efficacy on ADP induced platelet function may be influenced by concomitant antithrombotic therapies. Data regarding the effect of dabigatran on platelet function is limited to in vitro studies and healthy individuals. Methods. The “Dabi-ADP-1” and “Dabi-ADP-2” trials randomized patients with atrial fibrillation to either dabigatran or phenprocoumon for a 2-week period. In Dabi-ADP-1 (n = 70) patients with clopidogrel therapy were excluded and in Dabi-ADP-2 (n = 46) patients had to be treated concomitantly with clopidogrel. The primary endpoint was ADP-induced platelet aggregation between dabigatran and phenprocoumon at 14 days. Secondary endpoints were ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Results. There was no significant difference regarding the primary endpoint between both groups in either trial (Dabi-ADP-1: Dabigatran: 846 [650–983] AU × min versus phenprocoumon: 839 [666–1039] AU × min, P = 0.90 and Dabi-ADP-2: 326 [268–462] versus 350 [214–535], P = 0.70) or regarding the secondary endpoints, ADPtest HS-, TRAP-, and COL-induced platelet aggregation. Conclusion. Dabigatran as compared to phenprocoumon has no impact on ADP-induced platelet aggregation in atrial fibrillation patients neither with nor without concomitant clopidogrel therapy. PMID:26229963

  12. Targeting insulin amyloid assembly by small aromatic molecules: toward rational design of aggregation inhibitors.

    PubMed

    Levy-Sakin, Michal; Shreberk, Michal; Daniel, Yael; Gazit, Ehud

    2009-01-01

    Amyloid fibril formation is a common event in more than twenty human diseases and in some normal physiological processes. The mechanism of this ordered aggregation process and the molecular forces driving it are therefore of great importance. One of the strategies used in this field is targeting the fibrillization process by different factors, like, short peptides, organic molecules, etc. Here, we targeted insulin fibril formation by a range of small aromatic molecules, with different numbers of aromatic rings and various substituent groups. Using Thioflavin T fluorescence assay and transmission electron microscopy, we found that all dicyclic and tricyclic compounds in our screen were efficient inhibitors of insulin fibril formation. A common notion regarding amyloid inhibitors is that two functional groups are essentials for interfering with the amyloid formation process; a recognition motif and a bulky group for inducing a steric interference. However, here, we showed that some monocyclic compounds as small as toluene were also found to inhibit fibrillization. In addition, we found that substituent of benzene ring have a great influence on the inhibitory potency. Specifically, cyano, methyl and nitro groups increased the inhibitory potency. The results introduced here may contribute to future rational design of amyloid inhibitors.

  13. 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.

  14. Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro

    PubMed Central

    Dobrovolskaia, Marina A.; Patri, Anil K.; Simak, Jan; Hall, Jennifer B.; Semberova, Jana; De Paoli Lacerda, Silvia H.; McNeil, Scott E.

    2013-01-01

    Blood platelets are essential in maintaining hemostasis. Various materials can activate platelets and cause them to aggregate. Platelet aggregation in vitro is often used as a marker for materials’ thrombogenic properties and studying nanomaterial interaction with platelets is an important step toward understanding their hematocompatibility. Here we report evaluation of 12 formulations of PAMAM dendrimers varying in size and surface charge. Using a cell counter based method, light transmission aggregometry and scanning electron microscopy, we show that only large cationic dendrimers, but not anionic, neutral or small cationic dendrimers, induce aggregation of human platelets in plasma in vitro. The aggregation caused by large cationic dendrimers was proportional to the number of surface amines. The observed aggregation was not associated with membrane microparticle release, and was insensitive to a variety of chemical and biological inhibitors known to interfere with various pathways of platelet activation. Taken in context with previously reported studies, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation through disruption of membrane integrity. PMID:22026635

  15. 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

  16. A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells.

    PubMed

    Gandhi, Manish J; Drachman, Jonathan G; Reems, Jo-Anna; Thorning, David; Lannutti, Brian J

    2005-01-01

    Transfusion of allogeneic platelets is the mainstay of therapy for patients with thrombocytopenic hemorrhage. However, donated platelets can only be stored for 5 days and are maintained at room temperature, increasing the risk of bacterial growth. Developing a method to produce functional platelets in vitro would greatly advance transfusion therapy. During our studies to understand megakaryocyte development, we discovered that a Src kinase inhibitor, SU6656, induces cellular enlargement, polyploidization, and cytoplasmic fragmentation of several hematopoietic cell lines. Therefore, we tested the hypothesis that these fragments possess platelet-like activity. We studied a megakaryocytic cell-line, UT-7/TPO, and immature human primary megakaryocytes. After 6 days in the presence of thrombopoietin and SU6656, the majority of cells became polyploid and started shedding platelet-like fragments. These fragments were tested for aggregation and analyzed by electron microscopy. The platelet-like fragments did not undergo spontaneous activation but did show rapid and sustained aggregation in response to each of the standard agonists collagen, arachidonic acid, adenosine diphosphate, and epinephrine. Platelet-like fragments generated in SU6656 had higher amplitude and more prolonged aggregation in each of three experiments. Primary progenitors developed demarcation membranes within 72 h and evidence of dense granules and platelet-like fragments after 6 days. These cell fragments demonstrated properties consistent with platelet aggregation in response to multiple agonists without spontaneous aggregation. These studies provide evidence that SU6656 promotes megakaryocytic differentiation and thrombopoiesis in vitro. PMID:15923131

  17. 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

  18. 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. PMID:25730478

  19. 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.

  20. Engineering new bone via a minimally invasive route using human bone marrow-derived stromal cell aggregates, microceramic particles, and human platelet-rich plasma gel.

    PubMed

    Chatterjea, Anindita; Yuan, Huipin; Fennema, Eelco; Burer, Ruben; Chatterjea, Supriyo; Garritsen, Henk; Renard, Auke; van Blitterswijk, Clemens A; de Boer, Jan

    2013-02-01

    There is a rise in the popularity of arthroscopic procedures in orthopedics. However, the majority of cell-based bone tissue-engineered constructs (TECs) rely on solid preformed scaffolding materials, which require large incisions and extensive dissections for placement at the defect site. Thus, they are not suitable for minimally invasive techniques. The aim of this study was to develop a clinically relevant, easily moldable, bone TEC, amenable to minimally invasive techniques, using human mesenchymal stromal cells (hMSCs) and calcium phosphate microparticles in combination with an in situ forming platelet-rich plasma gel obtained from human platelets. Most conventional TECs rely on seeding and culturing single-cell suspensions of hMSCs on scaffolds. However, for generating TECs amenable to the minimally invasive approach, it was essential to aggregate the hMSCs in vitro before seeding them on the scaffolds as unaggregated MSCs did not generate any bone. Twenty four hours of in vitro aggregation was determined to be optimal for maintaining cell viability in vitro and bone formation in vivo. Moreover, no statistically significant difference was observed in the amount of bone formed when the TECs were implanted via an open approach or a minimally invasive route. TECs generated using MSCs from three different human donors generated new bone through the minimally invasive route in a reproducible manner, suggesting that these TECs could be a viable alternative to preformed scaffolds employed through an open surgery for treating bone defects.

  1. Correlation between spontaneous metastatic potential, platelet-aggregating activity of cell surface extracts, and cell surface sialylation in 10 metastatic-variant derivatives of a rat renal sarcoma cell line.

    PubMed Central

    Pearlstein, E; Salk, P L; Yogeeswaran, G; Karpatkin, S

    1980-01-01

    Several properties of 10 cell lines derived from the polyoma-induced PW20 Wistar-Furth rat renal sarcoma have been examined, including the ability of the tumor cells to metastasize spontaneously from subcutaneous sites in syngeneic hosts, the platelet-aggregating activity of material extracted by urea from the surface of cultured cells, the sialic acid content of the platelet-aggregating material, and the degree of sialylation of cell surface glycoconjugates in cultured cells. A correlation has been observed among all of these parameters. The results suggest a possible link between the degree of cell surface sialylation of tumor cells, their ability to aggregate platelets, and their ability to metastasize. PMID:6933486

  2. Thrombin-Mediated Platelet Activation of Lysed Whole Blood and Platelet-Rich Plasma: A Comparison Between Platelet Activation Markers and Ultrastructural Alterations.

    PubMed

    Augustine, Tanya N; van der Spuy, Wendy J; Kaberry, Lindsay L; Shayi, Millicent

    2016-06-01

    Platelet ultrastructural alterations representing spurious activation have been identified in pathological conditions. A limitation of platelet studies is that sample preparation may lead to artifactual activation processes which may confound results, impacting the use of scanning electron microscopy as a supplemental diagnostic tool. We used scanning electron microscopy and flow cytometry to analyze platelet activation in platelet-rich plasma (PRP) and whole blood (WB) samples. PRP generated using a single high g force centrifugation, and WB samples treated with a red blood cell lysis buffer, were exposed to increasing concentrations of the agonist thrombin. Platelets in lysed WB samples responded to thrombin by elevating the activation marker CD62p definitively, with corresponding ultrastructural changes indicating activation. Conversely, CD62p expression in PRP preparations remained static. Ultrastructural analysis revealed fully activated platelets even under low concentration thrombin stimulation, with considerable fibrin deposition. It is proposed that the method for PRP production induced premature platelet activation, preventable by using an inhibitor of platelet aggregation and fibrin polymerization. Nevertheless, our results show a definitive correspondence between flow cytometry and scanning electron microscopy in platelet activation studies, highlighting the potential of the latter technique as a supplemental diagnostic tool. PMID:27329313

  3. 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.

  4. Staphylococcus aureus α-toxin triggers the synthesis of B-cell lymphoma 3 by human platelets.

    PubMed

    Schubert, Sebastian; Schwertz, Hansjörg; Weyrich, Andrew S; Franks, Zechariah G; Lindemann, Stephan; Otto, Monika; Behr, Hagen; Loppnow, Harald; Schlitt, Axel; Russ, Martin; Presek, Peter; Werdan, Karl; Buerke, Michael

    2011-02-01

    The frequency and severity of bacteremic infections has increased over the last decade and bacterial endovascular infections (i.e., sepsis or endocarditis) are associated with high morbidity and mortality. Bacteria or secreted bacterial products modulate platelet function and, as a result, affect platelet accumulation at sites of vascular infection and inflammation. However, whether bacterial products regulate synthetic events in platelets is not known. In the present study, we determined if prolonged contact with staphylococcal α-toxin signals platelets to synthesize B-cell lymphoma (Bcl-3), a protein that regulates clot retraction in murine and human platelets. We show that α-toxin induced α(IIb)β(3)-dependent aggregation (EC(50) 2.98 µg/mL ± 0.64 µg/mL) and, over time, significantly altered platelet morphology and stimulated de novo accumulation of Bcl-3 protein in platelets. Adherence to collagen or fibrinogen also increased the expression of Bcl-3 protein by platelets. α-toxin altered Bcl-3 protein expression patterns in platelets adherent to collagen, but not fibrinogen. Pretreatment of platelets with inhibitors of protein synthesis or the mammalian Target of Rapamycin (mTOR) decreased Bcl-3 protein expression in α-toxin stimulated platelets. In conclusion, Staphylococcusaureus-derived α-toxin, a pore forming exotoxin, exerts immediate (i.e., aggregation) and prolonged (i.e., protein synthesis) responses in platelets, which may contribute to increased thrombotic events associated with gram-positive sepsis or endocarditis.

  5. Comparative in vitro and ex vivo activities of selected inhibitors of transthyretin aggregation: relevance in drug design

    PubMed Central

    Cardoso, Isabel; Almeida, Maria Rosário; Ferreira, Nelson; Arsequell, Gemma; Valencia, Gregorio; Saraiva, Maria João

    2007-01-01

    Destabilization of the tetrameric fold of TTR (transthyretin) is important for aggregation of the protein which culminates in amyloid fibril formation. Many TTR mutations interfere with tetramer stability, increasing the amyloidogenic potential of the protein. The vast majority of proposed TTR fibrillogenesis inhibitors are based on in vitro assays with isolated protein, limiting their future use in clinical assays. In the present study we investigated TTR fibrillogenesis inhibitors using a cellular system that produces TTR intermediates/aggregates in the medium. Plasmids carrying wild-type TTR, V30M or L55P cDNA were transfected into a rat Schwannoma cell line and TTR aggregates were investigated in the medium using a dot-blot filter assay followed by immunodetection. Results showed that, in 24 h, TTR L55P forms aggregates in the medium, whereas, up to 72 h, wild-type TTR and V30M do not. A series of 12 different compounds, described in the literature as in vitro TTR fibrillogenesis inhibitors, were tested for their ability to inhibit L55P aggregate formation; in this system, 2-[(3,5-dichlorophenyl) amino] benzoic acid, benzoxazole, 4-(3,5-difluorophenyl) benzoic acid and tri-iodophenol were the most effective inhibitors, as compared with the reference iododiflunisal, previously shown by ex vivo and in vitro procedures to stabilize TTR and inhibit fibrillogenesis. Among these drugs, 2-[(3,5-dichlorophenyl) amino] benzoic acid and tri-iodophenol stabilized TTR from heterozygotic carriers of V30M in the same ex vivo conditions as those used previously for iododiflunisal. The novel cellular-based test herein proposed for TTR fibrillogenesis inhibitor screens avoids not only lengthy and cumbersome large-scale protein isolation steps but also artefacts associated with most current in vitro first-line screening methods, such as those associated with acidic conditions and the absence of serum proteins. PMID:17683281

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

    SciTech Connect

    Given, M.B.

    1983-01-01

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

  7. 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

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-03-01

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

  10. Proteasome proteolysis supports stimulated platelet function and thrombosis

    PubMed Central

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

    2014-01-01

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

  11. 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

  12. Argan oil prevents prothrombotic complications by lowering lipid levels and platelet aggregation, enhancing oxidative status in dyslipidemic patients from the area of Rabat (Morocco)

    PubMed Central

    2013-01-01

    Background It is now established that patients with hyperlipidemia have a high risk of atherosclerosis and thrombotic complications, which are two important events responsible for the onset and progression of cardiovascular disease. In the context of managing dyslipidemia by means of dietary advice based on the consumption of argan oil, we wanted to investigate the effect of virgin argan oil on plasma lipids, and for the first time, on the platelet hyperactivation and oxidative status associated with dyslipidemia. This study concerns patients recruited in the area of Rabat in Morocco. Methods 39 dyslipidemic (79% women) patients were recruited for our study in the area of Rabat in Morocco. They were randomly assigned to the two following groups: the argan group, in which the subjects consumed 25 mL/day of argan oil at breakfast for 3 weeks, and the control group in which argan oil was replaced by butter. Results After a 3-week consumption period, blood total cholesterol was significantly lower in the argan oil group, as was LDL cholesterol (23.8% and 25.6% lower, respectively). However, the HDL cholesterol level had increased by 26% at the end of the intervention period compared to baseline. Interestingly, in the argan oil group thrombin-induced platelet aggregation was lower, and oxidative status was enhanced as a result of lower platelet MDA and higher GPx activity, respectively. Conclusions In conclusion, our results, even if it is not representative of the Moroccan population, show that argan oil can prevent the prothrombotic complications associated with dyslipidemia, which are a major risk factor for cardiovascular disease. PMID:23870174

  13. Nitridergic platelet pathway activation by hementerin, a metalloprotease from the leech Haementeria depressa.

    PubMed

    Chudzinski-Tavassi, Ana M; Bermej, Emilse; Rosenstein, Ruth E; Faria, Fernanda; Sarmiento, María I Keller; Alberto, Fabiana; Sampaio, Misako U; Lazzari, María A

    2003-09-01

    Hementerin (HT) is an 80 kDa fibrino(geno)lytic metalloprotease, purified from saliva of the leech Haementeria depressa. In the present report, the effect of HT on several functional parameters of human platelets was assessed. HT inhibited platelet aggregation and ATP release induced by different agonists such as ADP, adrenaline, collagen, thrombin, and arachidonic acid. HT did neither modify the expression of platelet glycoproteins (Ib, IIb-IIIa, Ia-IIa, IV) nor intraplatelet fibrinogen levels, whereas it markedly decreased CD62P and CD63 levels after the stimulation with thrombin. HT significantly increased thrombin-induced platelet Ca2+ intracellular levels, cGMP content and nitric oxide synthase (NOS) activity. The effect of HT on platelet aggregation was reversed by two NOS inhibitors, N(omega)-Nitro-L-arginine methyl ester and 2 N(G)-Nitro-L-arginine. In summary, these results indicate that HT is an effective inhibitor of human platelet aggregation, presumably through activation of the platelet's nitridergic pathway.

  14. First selective dual inhibitors of tau phosphorylation and Beta-amyloid aggregation, two major pathogenic mechanisms in Alzheimer's disease.

    PubMed

    Mariano, Marica; Schmitt, Christian; Miralinaghi, Parisa; Catto, Marco; Hartmann, Rolf W; Carotti, Angelo; Engel, Matthias

    2014-12-17

    In Alzheimer's disease (AD), multiple factors account for the accumulation of neurocellular changes, which may begin several years before symptoms appear. The most important pathogenic brain changes that are contributing to the development of AD are the formation of the cytotoxic β-amyloid aggregates and of the neurofibrillary tangles, which originate from amyloid-β peptides and hyperphosphorylated tau protein, respectively. New therapeutic agents that target both major pathogenic mechanisms may be particularly efficient. In this study, we introduce bis(hydroxyphenyl)-substituted thiophenes as a novel class of selective, dual inhibitors of the tau kinase Dyrk1A and of the amyloid-β aggregation. PMID:25247807

  15. Adhesion, activation, and aggregation of blood platelets and biofilm formation on the surfaces of titanium alloys Ti6Al4V and Ti6Al7Nb.

    PubMed

    Walkowiak-Przybyło, M; Klimek, L; Okrój, W; Jakubowski, W; Chwiłka, M; Czajka, A; Walkowiak, B

    2012-03-01

    Titanium alloys are still on the top list of fundamental materials intended for dental, orthopedics, neurological, and cardiovascular implantations. Recently, a special attention has been paid to vanadium-free titanium alloy, Ti6Al7Nb, that seems to represent higher biocompatibility than traditional Ti6Al4V alloy. Surprisingly, these data are not thoroughly elaborated in the literature; particularly there is a lack of comparative experiments conducted simultaneously and at the same conditions. Our study fills these shortcomings in the field of blood contact and microbiological colonization. To observe platelets adhesion and biofilm formation on the surfaces of compared titanium alloys, fluorescence microscope Olympus GX71 and scanning electron microscope HITACHI S-3000N were used. Additionally, flow cytometry analysis of platelets aggregation and activation in the whole blood after contact with sample surface, as an essential tool for biomaterial thrombocompatibility assessment, was proposed. As a result of our study it was demonstrated that polished surfaces of Ti6Al7Nb and Ti6Al4V alloys after contact with whole citrated blood and E. coli bacterial cells exhibit a considerable difference. Overall, it was established that Ti6Al4V has distinct tendency to higher thrombogenicity, more excessive bacterial biofilm formation and notable cytotoxic properties in comparison to Ti6Al7Nb. However, we suggest these studies should be extended for other types of cells and biological objects.

  16. Platelet Adhesion to Podoplanin Under Flow is Mediated by the Receptor CLEC-2 and Stabilised by Src/Syk-Dependent Platelet Signalling

    PubMed Central

    Pollitt, Alice Y.; Lowe, Kate; Latif, Arusa; Nash, Gerard B.

    2015-01-01

    Summary Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice. PMID:25694214

  17. Na+/H+ exchanger in regulation of platelet activation and paradoxical effects of cariporide

    PubMed Central

    Chang, He Benny; Gao, Xin; Nepomuceno, Rachel; Hu, Shaoshan; Sun, Dandan

    2015-01-01

    Platelets are anucleated cell fragments derived from mature megakaryocytes and function in hemostasis when the endothelium is injured. Hemostasis involving platelets can be divided into four phases: adhesion, activation, secretion, and aggregation. Platelet activation requires a rise in intracellular Ca2+ concentrations and results in both a morphological change and the secretion of platelet granule contents. Na+/H+ exchanger isoform 1 (NHE1) regulates the intracellular pH (pHi) and the volume of platelets. In addition, NHE1 plays a large role in platelet activation. Thrombus generation involves NHE1 activation and an increase in [Ca2+]i, which results from NHE1-mediated Na+ overload and the reversal of the Na+/Ca2+ exchanger. Cariporide (HOE-642), a potent NHE1 inhibitor, has inhibitory effects on the degranulation of human platelets, the formation of platelet–leukocyte-aggregates, and the activation of the GPIIb/IIIa receptor (PAC-1). However, despite the demonstrated protection of myocardial infarction as mediated by cariporide in patients undergoing coronary artery bypass graft surgery, the EXPEDITION clinical trial revealed that cariporide treatment increased mortality due to thromboembolic stroke. These findings suggest that a better understanding of NHE1 and its effect on platelet function and procoagulant factor regulation is warranted in order to develop therapies using NHE inhibitors. PMID:25595121

  18. 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

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

    PubMed

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

    2013-11-01

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

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

    PubMed

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

    2013-11-01

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

  1. Subpopulations in purified platelets adhering on glass.

    PubMed

    Donati, Alessia; Gupta, Swati; Reviakine, Ilya

    2016-01-01

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

  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. Enzymatic synthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, a hypotensive and platelet-aggregating lipid

    SciTech Connect

    Wykle, R.L.; Malone, B.; Snyder, F.

    1980-01-01

    1-Alkyl-2-acetyl-sn-glycero-3-phosphocholine, derived chemically from choline plasmalogens of beef heart, has been shown to possess powerful antihypertensive activity and to be an extremely potent platelet-activating factor. In the present study, microsomal preparations of rat spleen were shown to synthesize 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine by an acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase reaction; the acetyltransferase appears to be different from the acyltransferase responsible for the transfer of palmitate to glycerolipids.

  4. Proposal for an inhibitor of Alzheimer's disease blocking aggregation of amyloid-β peptides: ab initio molecular simulations

    NASA Astrophysics Data System (ADS)

    Okamoto, A.; Nomura, K.; Yano, A.; Higai, S.; Kondo, T.; Kamba, S.; Kurita, N.

    2013-04-01

    Aggregation of amyloid-β (Aβ) peptides is believed to play a key role in the mechanism of molecular pathogenesis of Alzheimer's disease (AD). To inhibit the aggregation and prevent AD, numerous compounds have been synthesized. A previous experimental study elucidated that a triazine derivative AA3E2 has anti-amyloidogenic ability, while a triazine derivative AA3D2 having a different substituent has no inhibitory effect. However, the reason for this remarkable difference in the ability cannot be explained by the chemical structures of these derivatives. In the present study, we present stable structures of the solvated complexes with Aβ and AA3E2/AA3D2 obtained by classical molecular mechanics method. The specific interactions between Aβ and AA3E2/AA3D2 in the complexes are investigated by ab initio fragment molecular orbital calculations. Based on the results obtained, we attempt to propose new potent inhibitors for the Aβ aggregation.

  5. 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.

  6. Complex disposition of methylthioninium redox forms determines efficacy in tau aggregation inhibitor therapy for Alzheimer's disease.

    PubMed

    Baddeley, Thomas C; McCaffrey, Jennifer; Storey, John M D; Cheung, John K S; Melis, Valeria; Horsley, David; Harrington, Charles R; Wischik, Claude M

    2015-01-01

    Methylthioninium (MT) is a tau aggregation inhibitor with therapeutic potential in Alzheimer's disease (AD). MT exists in equilibrium between reduced [leucomethylthioninium (LMT)] and oxidized (MT(+)) forms; as a chloride salt [methylthioninium chloride (MTC), "methylene blue"], it is stabilized in its MT(+) form. Although the results of a phase 2 study of MTC in 321 mild/moderate AD subjects identified a 138-mg MT/day dose as the minimum effective dose on cognitive and imaging end points, further clinical development of MT was delayed pending resolution of the unexpected lack of efficacy of the 228-mg MT/day dose. We hypothesized that the failure of dose response may depend on differences known at the time in dissolution in simulated gastric and intestinal fluids of the 100-mg MTC capsules used to deliver the 228-mg dose and reflect previously unsuspected differences in redox processing of MT at different levels in the gut. The synthesis of a novel chemical entity, LMTX (providing LMT in a stable anhydrous crystalline form), has enabled a systematic comparison of the pharmacokinetic properties of MTC and LMTX in preclinical and clinical studies. The quantity of MT released in water or gastric fluid within 60 minutes proved in retrospect to be an important determinant of clinical efficacy. A further factor was a dose-dependent limitation in the ability to absorb MT in the presence of food when delivered in the MT(+) form as MTC. A model is presented to account for the complexity of MT absorption, which may have relevance for other similar redox molecules. PMID:25320049

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

    PubMed

    Gleerup, G; Winther, K

    1995-08-01

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

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

    PubMed

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

    2014-06-01

    Purified active matrix metalloproteinase-2 (MMP-2) is able to promote platelet aggregation. We aimed to assess the role of MMP-2 expressed in atherosclerotic plaques in the platelet-activating potential of human carotid plaques and its correlation with ischaemic events. Carotid plaques from 81 patients undergoing endarterectomy were tested for pro-MMP-2 and TIMP-2 content by zymography and ELISA. Plaque extracts were incubated with gel-filtered platelets from healthy volunteers for 2 minutes before the addition of a subthreshold concentration of thrombin receptor activating peptide-6 (TRAP-6) and aggregation was assessed. Moreover, platelet deposition on plaque extracts immobilised on plastic coverslips under high shear-rate flow conditions was measured. Forty-three plaque extracts (53%) potentiated platelet aggregation (+233 ± 26.8%), an effect prevented by three different specific MMP-2 inhibitors (inhibitor II, TIMP-2, moAb anti-MMP-2). The pro-MMP-2/TIMP-2 ratio of plaques potentiating platelet aggregation was significantly higher than that of plaques not potentiating it (3.67 ± 1.21 vs 1.01 ± 0.43, p<0.05). Moreover, the platelet aggregation-potentiating effect, the active-MMP-2 content and the active MMP-2/pro-MMP-2 ratio of plaque extracts were significantly higher in plaques from patients who developed a subsequent major cardiovascular event. In conclusion, atherosclerotic plaques exert a prothrombotic effect by potentiating platelet activation due to their content of MMP-2; an elevated MMP-2 activity in plaques is associated with a higher rate of subsequent ischaemic cerebrovascular events. PMID:24499865

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

    PubMed Central

    2011-01-01

    Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml) in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM). Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM)-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA) inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLC)γ2 phosphorylation, protein kinase C (PKC) activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may have a great impact when

  10. 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

  11. [THE REFERENCE VALUES OF AGGREGATION OF PLATELETS IN ADULT POPULATION OF THE ASTRAKHAN OBLAST USING AGGREGOMETER MULTIPLATE].

    PubMed

    Petrova, O V; Shashin, S A; Tarasov, D G; Jukova, E R; Panova, E V; Gracheva, N P

    2016-01-01

    The modern international standards recommend each laboratory to develop or to confirm available in literature the reference intervalsfor every laboratory indicator In the Astrakhanskaia oblast, sampling of128 healthy males andfemales were examinedfor aggregation function of thrombocytes using impedance technique and applying aggregometer Multiplate ("Verum Diagnostica", Germany). The study used as inductors peptide activating receptor of thrombin; arachidonic and adenosine diphosphoric acids. The reference range of aggregation of thrombocytes with peptide activating receptor of thrombin, at aggregometer Multiplate, in healthy population of theAstrakhanskaia oblast made up to 815.2-1498.4 AU/min, with arachidonic acid--660-1341 AU/min. with adenosine diphosphoric acid--598-1120 AU/min. PMID:27183729

  12. 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

  13. The role of adenosine triphosphate citrate lyase in the metabolism of acetyl coenzyme a and function of blood platelets in diabetes mellitus.

    PubMed

    Michno, Anna; Skibowska, Anna; Raszeja-Specht, Anna; Cwikowska, Justyna; Szutowicz, Andrzej

    2004-01-01

    Diabetes is known to increase blood platelet activity. Activities of pyruvate dehydrogenase (PDH), adenosine triphosphate (ATP)-citrate lyase (ATPCL), acetyl-coenzyme A (acetyl-CoA) content, malonyl dialdehyde (MDA), synthesis, and platelet aggregation in resting conditions and after activation with thrombin were measured in diabetic subjects and in age- and sex-matched healthy subjects. Activities of ATPCL and PDH, acetyl-CoA content, and thrombin-evoked MDA synthesis as well as platelet aggregation in diabetes were 31%, 51%, 62%, 35%, and 21%, respectively, higher than in healthy subjects. In addition, activation of diabetic platelets caused 2 times greater release of acetyl-CoA from their mitochondria than in controls. Both 1.0 mmol/L (-)hydroxycitrate and 0.1 mmol/L SB-204490 decreased acetyl-CoA content in platelet cytoplasm along with suppression of MDA synthesis and platelet aggregation. These inhibitory effects were about 2 times greater in diabetic than in control platelets. The data presented indicate that the ATPCL pathway is operative in human platelets and may be responsible for provision of about 50% of acetyl units from their mitochondrial to cytoplasmic compartment. Increased acetyl-CoA synthesis in diabetic platelets may be the cause of their excessive activity in the course of the disease. ATPCL may be a target for its specific inhibitors as factors decreasing platelet activity. PMID:14681844

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

    PubMed

    Maheswaraiah, Anikisetty; Rao, Lingamallu Jaganmohan; Naidu, Kamatham Akhilender

    2015-03-01

    Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders.

  15. Full activation of mouse platelets requires ADP secretion regulated by SERCA3 ATPase-dependent calcium stores.

    PubMed

    Elaïb, Ziane; Adam, Frédéric; Berrou, Eliane; Bordet, Jean-Claude; Prévost, Nicolas; Bobe, Régis; Bryckaert, Marijke; Rosa, Jean-Philippe

    2016-08-25

    The role of the sarco-endoplasmic reticulum calcium (Ca(2+)) adenosine triphosphatase (ATPase) 3 (SERCA3) in platelet physiology remains poorly understood. Here, we show that SERCA3 knockout (SERCA3(-/-)) mice exhibit prolonged tail bleeding time and rebleeding. Thrombus formation was delayed both in arteries and venules in an in vivo ferric chloride-induced thrombosis model. Defective platelet adhesion and thrombus growth over collagen was confirmed in vitro. Adenosine 5'-diphosphate (ADP) removal by apyrase diminished adhesion and thrombus growth of control platelets to the level of SERCA3(-/-) platelets. Aggregation, dense granule secretion, and Ca(2+) mobilization of SERCA3(-/-) platelets induced by low collagen or low thrombin concentration were weaker than controls. Accordingly, SERCA3(-/-) platelets exhibited a partial defect in total stored Ca(2+) and in Ca(2+) store reuptake following thrombin stimulation. Importantly ADP, but not serotonin, rescued aggregation, secretion, and Ca(2+) mobilization in SERCA3(-/-) platelets, suggesting specificity. Dense granules appeared normal upon electron microscopy, mepacrine staining, and total serotonin content, ruling out a dense granule defect. ADP induced normal platelet aggregation, excluding a defect in ADP activation pathways. The SERCA3-specific inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone diminished both Ca(2+) mobilization and secretion of control platelets, as opposed to the SERCA2b inhibitor thapsigargin. This confirmed the specific role of catalytically active SERCA3 in ADP secretion. Accordingly, SERCA3-dependent Ca(2+) stores appeared depleted in SERCA3(-/-) platelets. Finally, αIIbβ3 integrin blockade did not affect SERCA3-dependent secretion, therefore proving independent of αIIbβ3 engagement. Altogether, these results show that SERCA3-dependent Ca(2+) stores control a specific ADP secretion pathway required for full platelet secretion induced by agonists at low concentration and independent

  16. Effects of green tea or Sasa quelpaertensis bamboo leaves on plasma and liver lipids, erythrocyte Na efflux, and platelet aggregation in ovariectomized rats.

    PubMed

    Ryou, Sung Hee; Kang, Min Sook; Kim, Kyu Il; Kang, Young Hee; Kang, Jung Sook

    2012-04-01

    This study was conducted to investigate the effects of Sasa quelpaertensis bamboo and green tea on plasma and liver lipids, platelet aggregation, and erythrocyte membrane Na channels in ovariectomized (OVX) rats. Thirty female rats were OVX, and ten female rats were sham-operated at the age of 6 weeks. The rats were divided into four groups at the age of 10 weeks and fed the experiment diets: sham-control, OVX-control, OVX-bamboo leaves (10%), or OVX-green tea leaves (10%) for four weeks. Final body weight increased significantly in the OVX groups compared with that in the sham-control, whereas body weight in the OVX-green tea group decreased significantly compared with that in the OVX-control (P < 0.01). High density lipoprotein (HDL)-cholesterol level decreased in all OVX groups compared with that in the sham-control rats (P < 0.05) but without a difference in plasma total cholesterol. Plasma triglycerides in the OVX-green tea group were significantly lower than those in the sham-control or OVX-control group (P < 0.05). Liver triglycerides increased significantly in the OVX-control compared with those in the sham-control (P < 0.01) but decreased significantly in the OVX-green tea group compared with those in the OVX-control or OVX-bamboo group (P < 0.01). Platelet aggregation in both maximum and initial slope tended to be lower in all OVX rats compared with that in the sham-control rats but was not significantly different. Na-K ATPase tended to increase and Na-K cotransport tended to decrease following ovariectomy. Na-K ATPase decreased significantly in the OVX-green tea group compared with that in the OVX-control group (P < 0.01), and Na-K cotransport increased significantly in the OVX-bamboo and OVX-green tea groups compared with that in the OVX-control (P < 0.05). Femoral bone mineral density tended to be lower in OVX rats than that in the sham-control, whereas the green tea and bamboo leaves groups recovered bone density to some extent. The results show that

  17. Platelet function defects in chronic alcoholism.

    PubMed Central

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

    1986-01-01

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

  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. Syntheses and evaluation of novel isoliquiritigenin derivatives as potential dual inhibitors for amyloid-beta aggregation and 5-lipoxygenase.

    PubMed

    Chen, Yi-Ping; Zhang, Zi-Ying; Li, Yan-Ping; Li, Ding; Huang, Shi-Liang; Gu, Lian-Quan; Xu, Jun; Huang, Zhi-Shu

    2013-08-01

    A series of new isoliquiritigenin (ISL) derivatives were synthesized and evaluated as dual inhibitors for amyloid-beta (Aβ) aggregation and 5-lipoxygenase (5-LO). It was found that all these synthetic compounds inhibited Aβ (1-42) aggregation effectively with their IC₅₀ values ranged from 2.2 ± 1.5 μM to 23.8 ± 2.0 μM. These derivatives also showed inhibitory activity to 5-LO with their IC50 values ranged from 6.1 ± 0.1 μM to 35.9 ± 0.3 μM. Their structure-activity relationships (SAR) and mechanisms of inhibitions were studied. This study provided potentially important information for further development of ISL derivatives as multifunctional agents for Alzheimer's disease (AD) treatment.

  20. Two nitrogen-containing ligands as inhibitors of metal-induced amyloid β-peptide aggregation.

    PubMed

    Chen, Tingting; Zhu, Shajun; Liu, Siyuan; Lu, Yapeng; Zhu, Li

    2014-02-01

    Abnormal interactions of Zn(2+) and Cu(2+) with the amyloid β-peptide (Aβ) are proposed to play an important role in the neuropathogenesis of Alzheimer's disease (AD). Metal chelators are potential therapeutic agents for AD because they could sequester metals ions from Aβ aggregates and reverse the aggregation. In this study, two nitrogencontaining ligands, TACN and BPA, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. The interactions between the chelators and Aβ40 aggregates are studied by turbidometry, thioflavin T (ThT) fluorescence spectroscopy, inductively coupled plasma mass spectrometry (ICP-MS), BCA protein assay, circular dichroism spectroscopy (CD), and atomic force microscopy (AFM). The results demonstrates that TACN and BPA are capable of both disrupting and preventing Zn(2+) or Cu(2+)-induced Aβ40 aggregation. Moreover, they can also suppress the production of H2O2 induced by Cu-Aβ40, associated with toxic oxidative stress in AD. PMID:23844690

  1. Specific inhibitory effects of the NO donor MAHMA/NONOate on human platelets.

    PubMed

    Kobsar, Anna; Simonis, Sandra; Klinker, Erdwine; Koessler, Angela; Kuhn, Sabine; Boeck, Markus; Koessler, Juergen

    2014-07-15

    Nitric oxide (NO) is a physiological inhibitor of platelet function and has vaso-dilating effects. Therefore, synthesized NO releasing agents are used e.g. in cardiovascular medicine. The aim of this study was to characterise specific effects of the short living agent MAHMA/NONOate, a NO donor of the diazeniumdiolate class, on human platelets. Whole blood was obtained from healthy volunteers. In washed human platelets, the MAHMA/NONOate induced phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and cyclic nucleotide production were studied by Western Blot and by enzyme immunoassay kits. Agonist induced aggregation was measured in platelet rich plasma. Paired Student׳s t-test was used for statistical analysis. MAHMA/NONOate significantly stimulated platelet VASP phosphorylation in a concentration dependent manner and increased intracellular cGMP, but not cAMP levels, transiently. ODQ, a specific inhibitor of the soluble guanylyl cyclase, completely prevented VASP phosphorylation induced by low MAHMA/NONOate concentrations (5nM-15nM). The effects of higher concentrations (30-200nM) were only partially inhibited by ODQ. MAHMA/NONOate reduced platelet aggregation induced by low doses of agonists (2µM ADP, 0.5µg/mL collagen, 5µM TRAP-6) in a concentration dependent manner. MAHMA/NONOate leads to a rapid and transient activation of platelet inhibitory systems, accompanied by decreased platelet aggregation induced by low dose agonists. At low MAHMA/NONOate concentrations, the effects are cGMP dependent and at higher concentrations additionally cGMP independent. The substance could be of interest for clinical situations requiring transient and subtotal inhibition of platelet function. PMID:24780647

  2. A randomised determination of the Effect of Fluvastatin and Atorvastatin on top of dual antiplatelet treatment on platelet aggregation after implantation of coronary drug-eluting stents. The EFA-Trial.

    PubMed

    Wenaweser, Peter; Eshtehardi, Parham; Abrecht, Linda; Zwahlen, Marcel; Schmidlin, Kurt; Windecker, Stephan; Meier, Bernhard; Haeberli, Andre; Hess, Otto M

    2010-09-01

    Drug-drug interaction between statins metabolised by cytochrome P450 3A4 and clopidogrel have been claimed to attenuate the inhibitory effect of clopidogrel. However, published data regarding this drug-drug interaction are controversial. We aimed to determine the effect of fluvastatin and atorvastatin on the inhibitory effect of dual antiplatelet therapy with acetylsalicylic acid (ASA) and clopidogrel. One hundred one patients with symptomatic stable coronary artery disease undergoing percutaneous coronary intervention and drug-eluting stent implantation were enrolled in this prospective randomised study. After an interval of two weeks under dual antiplatelet therapy with ASA and clopidogrel, without any lipid-lowering drug, 87 patients were randomised to receive a treatment with either fluvastatin 80 mg daily or atorvastatin 40 mg daily in addition to the dual antiplatelet therapy for one month. Platelet aggregation was assessed using light transmission aggregometry and whole blood impedance platelet aggregometry prior to randomisation and after one month of receiving assigned statin and dual antiplatelet treatment. Platelet function assessment after one month of statin and dual antiplatelet therapy did not show a significant change in platelet aggregation from 1st to 2nd assessment for either statin group. There was also no difference between atorvastatin and fluvastatin treatment arms. In conclusion, neither atorvastatin 40 mg daily nor fluvastatin 80 mg daily administered in combination with standard dual antiplatelet therapy following coronary drug-eluting stent implantation significantly interfere with the antiaggregatory effect of ASA and clopidogrel.

  3. Isolation, functional characterization and proteomic identification of CC2-PLA₂ from Cerastes cerastes venom: a basic platelet-aggregation-inhibiting factor.

    PubMed

    Chérifi, Fatah; Namane, Abdelkader; Laraba-Djebari, Fatima

    2014-02-01

    Three-step chromatography and proteomic analysis have been used to purify and characterize a new basic phospholipase A₂ named CC2-PLA₂ from the venom of Cerastes cerastes. This phospholipase A₂ has been isolated to an extent of about 50-folds and its molecular weight was estimated at 13,534 Da. For CC2-PLA₂ identification and LC-MALDI-MS/MS analysis, the protein was reduced, alkylated and double hydrolyzed by lysine-C endopeptidase and trypsin. Tryptic fragments of LC-MS/MS analyzed CC2-PLA₂ showed sequence similarities with other snake venom PLA₂. This presents only 51 % (61/120 amino acid residues) sequence homology with the first PLA₂ (gi |129506|) previously purified from the same venom. The isolated CC2-PLA₂ displayed anti-aggregative effect on platelets and induced an inflammatory response characterized by leukocytosis in the peripheral blood. This inflammatory response is accompanied by a release of inflammatory mediators such as IL-6, eosinophil peroxidase and complement system. Obtained results indicate that CC2-PLA₂ induced a release of high level of pro-inflammatory (IL-6) cytokine and no effect on the level of anti-inflammatory cytokine (IL-10) in blood sera. Furthermore, eosinophil peroxidase activity and hemolytic complement effect increased in peripheral blood. Mononuclear and neutrophil cells were found predominant in the induced leucocytosis following CC2-PLA₂ administration into animals.

  4. Higher levels of circulating monocyte-platelet aggregates are correlated with viremia and increased sCD163 levels in HIV-1 infection.

    PubMed

    Liang, Hua; Duan, Zhaojun; Li, Dan; Li, Dongliang; Wang, Zheng; Ren, Li; Shen, Tao; Shao, Yiming

    2015-07-01

    Increased levels of monocyte-platelet aggregates (MPAs) are reported to be highly correlated with cardiovascular events. In this study, the MPA levels in different monocyte subsets and the associations between MPA levels, HIV-1 viremia and monocyte activation were evaluated during HIV-1 infection. The results showed that the percentages of MPAs in all three monocyte subsets were higher in HIV-1-infected subjects than in healthy controls, and were associated with the plasma viral load in the non-classical and intermediate monocyte subsets. The plasma levels of sCD14 and sCD163 were upregulated in HIV-1 infection and were positively associated with viral loads and negatively associated with CD4 counts. P-selectin glycoprotein ligand-1 (PSGL-1) was shown to be expressed at significantly lower levels on all three monocyte subsets and was negatively correlated with the sCD163 level. The MPA level was correlated with the levels of plasma sCD163 but negatively correlated with CD163 and PSGL-1 on all three monocyte subsets. An elevated immune activation status was correlated with increased MPA formation, underlying the potential interaction between monocyte activation and MPA formation. This interaction may be related to a higher thromboembolic risk in patients infected with HIV-1.Cellular & Molecular Immunology advance online publication, 11 August 2014; doi:10.1038/cmi.2014.66.

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

    PubMed

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

    2010-01-01

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

  6. Purification and characterization of AHPM, a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities, from Agkistrodon halys pallas venom.

    PubMed

    Song, Jiajia; Xu, Xiaolong; Zhang, Yan; Guo, Mingchun; Yan, Xincheng; Wang, Shasha; Gao, Shang

    2013-04-01

    A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110 kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn(2+) and Ca(2+) ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn(2+) ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200 ± 8 nM and 280 ± 10 nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30 μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent. PMID:23104267

  7. Aprotinin inhibits the contact, neutrophil, and platelet activation systems during simulated extracorporeal perfusion.

    PubMed

    Wachtfogel, Y T; Kucich, U; Hack, C E; Gluszko, P; Niewiarowski, S; Colman, R W; Edmunds, L H

    1993-07-01

    Aprotinin reduces blood loss after cardiac operations and decreases the bleeding time. The mechanism of action of aprotinin that produces these effects is not clear. During simulated extracorporeal circulation the contact and complement systems, platelets, and neutrophils are activated. We investigated the effect of aprotinin on kallikrein-C1-inhibitor complex and C1-C1-inhibitor complex formation, neutrophil degranulation, and platelet release and aggregation during simulated extracorporeal circulation. Fresh heparinized human blood was recirculated at 37 degrees C for 2 hours in a spiral coil membrane oxygenator-roller pump perfusion circuit. Changes in platelet count, leukocyte count, platelet response to adenosine diphosphate, and plasma levels of beta-thromboglobulin, kallikrein-C1-inhibitor complexes, C1-C1-inhibitor complexes, and neutrophil elastase were measured before and at 5, 30, 60, and 120 minutes of recirculation at 0, 0.015, 0.03, 0.06, and 0.12 mg/ml doses of aprotinin. Platelet counts decreased to 36% +/- 12% of control values at 5 minutes and increased to 56% +/- 13% at 120 minutes without aprotinin. Aprotinin did not affect platelet counts, but it did prevent the decrease in sensitivity of platelets to adenosine diphosphate and it attenuated beta-thromboglobulin release. In the absence of aprotinin, kallikrein-C1-inhibitor and C1-C1-inhibitor complexes increased progressively to 0.53 +/- 0.14 U/ml and 2.38 +/- 0.33 U/ml, respectively, at 120 minutes. Kallikrein-C1-inhibitor complexes were completely inhibited and C1-C1-inhibitor complexes were partially inhibited at aprotinin concentrations of 0.03 mg/ml or greater. Release of neutrophil elastase was partially but not completely inhibited at the highest dose of aprotinin and was 50% inhibited at a dose of 0.03 mg/ml. Because activation of the fibrinolytic system does not occur in this system, the changes were independent of the inhibition of plasmin. We conclude that aprotinin in high doses

  8. Schistosomes versus platelets.

    PubMed

    Da'dara, Akram A; Skelly, Patrick J

    2014-12-01

    Schistosomes are parasitic platyhelminths that currently infect >200million people and cause the chronic debilitating disease schistosomiasis. While these large intravascular parasites can disturb blood flow, they do not appear to activate platelets and provoke thrombus formation. Host-interactive tegumental molecules have been proposed to be important in this regard. For example, tegumental apyrase, SmATPDase1 can degrade the platelet-activating molecule ADP in the extracellular environment. The parasites themselves can produce prostaglandins (or may induce prostaglandin production by host cells) which could inhibit platelet aggregation. Additional tegumental proteins have been proposed to impede the coagulation cascade and to promote fibrinolysis. Platelets have been shown to be directly toxic to schistosomes. Platelets recovered from infected rats are able to kill larval parasites in culture and platelets obtained at later times post-infection are generally better at killing. Even platelets from uninfected rats can rapidly kill larval schistosomes if first exposed to a variety of activators (such as: serum from infected rats, the IgE fraction of that serum, C-reactive protein, cytokines (TNFα or TNFβ)). Passive transfer of stimulated platelets can protect rats against a challenge schistosome infection. Cytokines (TNFα, TNFβ, IFNγ or IL-6) have been shown to similarly promote normal human platelet killing of schistosomes in vitro. Platelet antimicrobial effector molecules (e.g. platelet microbicidal proteins) may mediate such killing. While platelets can be protective against schistosomes following infection of humans and mice, platelet numbers decline (but not so in the non-permissive rat host) and coagulopathy becomes more apparent as schistosome-induced pathology increases.

  9. Relative activities on and uptake by human blood platelets of 5-hydroxytryptamine and several analogues

    PubMed Central

    Born, G. V. R.; Juengjaroen, Kanchana; Michal, F.

    1972-01-01

    1. The specificity of platelet receptor sites for 5-HT uptake and for the rapid morphological change and aggregation was investigated with 5-hydroxy-tryptamine (5-HT) and seventeen analogues as well as with some antagonists of 5-HT. 2. The analogues, with the exception of 5-hydroxy-N'N'-dibutyltryptamine, caused the rapid morphological change in platelets. In concentrations below those needed to produce the agonistic action (viz. 0.05-2.0 μM), these analogues themselves inhibited competitively the shape change caused by 5-HT. 3. The velocity of change in shape caused by 5-HT was reduced in low Na media. 4. Ten analogues produced platelet aggregation; three of these, viz. 5-methoxy-α-methyltryptamine, 5-hydroxy-α-methyltryptamine and 5-hydroxy-N'N'-diisopropyltryptamine), were approximately equipotent with 5-HT. Six analogues did not induce platelet aggregation. 5. All the analogues which prevented the initial change in shape of platelets caused by 5-HT also inhibited its aggregating effect, apparently competitively with low Ki values (0.02-1.6 μM). 6. As with the inhibition of shape change, the inhibition of aggregation shows relatively low structural specificity of the receptor site. 7. Methysergide was a potent inhibitor of shape change and aggregation (Ki∼0.03 μM); imipramine was much less inhibitory (Ki∼5-10 μM). 8. Only one analogue (5-hydroxy-α-methyltryptamine) was taken up like 5-HT by platelets. All the other analogues inhibited the uptake of 5-HT by platelets (Ki=0.2-2.7 μM). 9. Methysergide was a weak inhibitor of 5-HT uptake (Ki∼125 μM) whereas imipramine was very effective (Ki∼0.3 μM). 10. Our results show that the initial change in shape of platelets is required for and precedes aggregation. The structural specificity of the platelet receptor concerned with shape change and aggregation caused by 5-HT appears low whereas the uptake mechanism is a highly specific one. The uptake probably proceeds through more than one step, the

  10. Augmentation of platelet and endothelial cell eNOS activity decreases sepsis-related neutrophil-endothelial cell interactions.

    PubMed

    Khan, Raymond; Kirschenbaum, Linda A; LaRow, Catherine; Berna, Gioiamaria; Griffin, Kelly; Astiz, Mark E

    2010-03-01

    NO is an important mediator of microvascular patency and blood flow. The purpose of this study was to examine the role of enhanced eNOS activity in attenuating sepsis-induced neutrophil-endothelial cell interactions. Microslides coated with human umbilical vein endothelial cells were stimulated with plasma from patients with septic shock. Neutrophil and platelets from control subjects were also stimulated with plasma from patients in septic shock and perfused over stimulated endothelial cells. l-Arginine (LA) with and without NG-monomethyl l-arginine (LNMMA), a nonselective NOS inhibitor, and N-(3-(aminomethyl) benzyl acetamide) ethanimidamide dihydrochloride (1400W), a highly selective iNOS inhibitor, were added to the septic plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophil aggregates were determined. Cell activation and the formation of platelet-neutrophil aggregates were assessed by flow cytometry. Separate experiments were done with isolated platelets using platelet aggregometry. l-Arginine significantly decreased sepsis-related neutrophil adhesion and aggregation and increased rolling velocity. The addition of LNMMA to LA and cell suspensions reversed the effects of LA on these parameters, whereas the addition of 1400W had no effect on LA-related changes. Platelet-neutrophil aggregation, platelet aggregation, platelet activation, and neutrophil activation induced by septic plasma were also significantly decreased by LA. Again, the addition of LNMMA reversed the effects of LA on these parameters, whereas 1400W had no effect on LA-related changes. These data suggest that enhancement of platelet and endothelial cell eNOS activity decreases sepsis-induced neutrophil-endothelial cell interactions and may play a role in maintaining microvascular patency in septic shock.

  11. Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels in vivo and platelet aggregation in vitro

    PubMed Central

    Yuvaraju, Priya; Beegam, Sumaya; Ali, Badreldin H

    2015-01-01

    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. PMID:25662827

  12. 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.

  13. Thrombin Receptor-Activating Protein (TRAP)-Activated Akt Is Involved in the Release of Phosphorylated-HSP27 (HSPB1) from Platelets in DM Patients

    PubMed Central

    Tokuda, Haruhiko; Kuroyanagi, Gen; Tsujimoto, Masanori; Matsushima-Nishiwaki, Rie; Akamatsu, Shigeru; Enomoto, Yukiko; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

    2016-01-01

    It is generally known that heat shock protein 27 (HSP27) is phosphorylated through p38 mitogen-activated protein (MAP) kinase. We have previously reported that HSP27 is released from human platelets associated with collagen-induced phosphorylation. In the present study, we conducted an investigation into the effect of thrombin receptor-activating protein (TRAP) on the release of HSP27 in platelets in type 2 diabetes mellitus (DM) patients. The phosphorylated-HSP27 levels induced by TRAP were directly proportional to the aggregation of platelets. The levels of phosphorylated-HSP27 (Ser-78) were correlated with the levels of phosphorylated-p38 MAP kinase and phosphorylated-Akt in the platelets stimulated by 10 µM TRAP but not with those of phosphorylated-p44/p42 MAP kinase. The levels of HSP27 released from the TRAP (10 µM)-stimulated platelets were correlated with the levels of phosphorylated-HSP27 in the platelets. The released platelet-derived growth factor-AB (PDGF-AB) levels were in parallel with the HSP27 levels released from the platelets stimulated by 10 µM TRAP. Although the area under the curve (AUC) of small aggregates (9–25 µm) induced by 10 µM TRAP showed no significant correlation with the released HSP27 levels, AUC of medium aggregates (25–50 µm), large aggregates (50–70 µm) and light transmittance were significantly correlated with the released HSP27 levels. TRAP-induced phosphorylation of HSP27 was truly suppressed by deguelin, an inhibitor of Akt, in the platelets from a healthy subject. These results strongly suggest that TRAP-induced activation of Akt in addition to p38 MAP kinase positively regulates the release of phosphorylated-HSP27 from human platelets, which is closely related to the platelet hyper-aggregation in type 2 DM patients. PMID:27187380

  14. Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo

    PubMed Central

    Momi, Stefania; Falcinelli, Emanuela; Giannini, Silvia; Ruggeri, Loredana; Cecchetti, Luca; Corazzi, Teresa; Libert, Claude

    2009-01-01

    Platelet activation at a site of vascular injury is essential for the arrest of bleeding; however, excessive platelet activation at a site of arterial damage can result in the unwarranted formation of arterial thrombi, precipitating acute myocardial infarction, or ischemic stroke. Activation of platelets beyond the purpose of hemostasis may occur when substances facilitating thrombus growth and stability accumulate. Human platelets contain matrix metalloproteinase 2 (MMP-2) and release it upon activation. Active MMP-2 amplifies the platelet aggregation response to several agonists by potentiating phosphatidylinositol 3-kinase activation. Using several in vivo thrombosis models, we show that the inactivation of the MMP-2 gene prevented thrombosis induced by weak, but not strong, stimuli in mice but produced only a moderate prolongation of the bleeding time. Moreover, using cross-transfusion experiments and wild-type/MMP-2−/− chimeric mice, we show that it is platelet-derived MMP-2 that facilitates thrombus formation. Finally, we show that platelets activated by a mild vascular damage induce thrombus formation at a downstream arterial injury site by releasing MMP-2. Thus, platelet-derived MMP-2 plays a crucial role in thrombus formation by amplifying the response of platelets to weak activating stimuli. These findings open new possibilities for the prevention of thrombosis by the development of MMP-2 inhibitors. PMID:19808257

  15. Piceatannol and Other Wine Stilbenes: A Pool of Inhibitors against α-Synuclein Aggregation and Cytotoxicity

    PubMed Central

    Temsamani, Hamza; Krisa, Stéphanie; Decossas-Mendoza, Marion; Lambert, Olivier; Mérillon, Jean-Michel; Richard, Tristan

    2016-01-01

    The aggregation of α-synuclein is one on the key pathogenic events in Parkinson’s disease. In the present study, we investigated the inhibitory capacities of stilbenes against α-synuclein aggregation and toxicity. Thioflavin T fluorescence, transmission electronic microscopy, and SDS-PAGE analysis were performed to investigate the inhibitory effects of three stilbenes against α-synuclein aggregation: piceatannol, ampelopsin A, and isohopeaphenol. Lipid vesicle permeabilization assays were performed to screen stilbenes for protection against membrane damage induced by aggregated α-synuclein. The viability of PC12 cells was examined using an MTT assay to assess the preventive effects of stilbenes against α-synuclein-induced toxicity. Piceatannol inhibited the formation of α synuclein fibrils and was able to destabilize preformed filaments. It seems to induce the formation of small soluble complexes protecting membranes against α-synuclein-induced damage. Finally, piceatannol protected cells against α-synuclein-induced toxicity. The oligomers tested (ampelopsin A and hopeaphenol) were less active. PMID:27314384

  16. Endotoxemia alters nucleotide hydrolysis in platelets of rats.

    PubMed

    Vuaden, Fernanda Cenci; Furstenau, Cristina Ribas; Savio, Luiz Eduardo Baggio; Sarkis, João José Freitas; Bonan, Carla Denise

    2009-03-01

    Platelets play a critical role in homeostasis and blood clotting at sites of vascular injury, and also in various ways in innate immunity and inflammation. Platelets are one of the first cells to accumulate at an injured site, and local release of their secretome at some point initiate an inflammatory cascade that attracts leukocytes, activates target cells, stimulates vessel growth and repair. The level of exogenous ATP in the body may be increased in various inflammatory and shock conditions, primarily as a consequence of nucleotide release from platelets, endothelium and blood vessel cells. An increase of ATP release has been described during inflammation and this compound presents proinflammatory properties. ADP is a nucleotide known to induce changes in platelets shape and aggregation, to promote the exposure of fibrinogen-binding sites and to inhibit the stimulation of adenylate cyclase. Adenosine, the final product of the nucleotide hydrolysis, is a vasodilator and an inhibitor of platelet aggregation. There is a group of ecto-enzymes responsible for extracellular nucleotide hydrolysis named ectonucleotidases, which includes the NTPDase (nucleoside triphosphate diphosphohydrolase) family, the NPP (nucleoside pyrophosphatase/phosphodiesterase) family and an ecto-5'-nucleotidase. Therefore, we have aimed to investigate the effect of lipopolysaccharide endotoxin from Escherichia coli on ectonucleotidases in platelets from adult rats in order to better understand the role of extracellular adenine nucleotides and nucleosides in the maintenance of blood homeostasis in inflammatory processes. LPS administered in vitro was not able to alter the ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis of platelets from untreated rats in all concentrations tested (25-100 microg/ml). There was a significant decrease in ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis in rat platelets after 48 hours of LPS exposure (2 mg/Kg, i.p.). ATP and ADP hydrolysis has been reduced about 28

  17. Nutritional zinc increases platelet reactivity.

    PubMed

    Marx, G; Krugliak, J; Shaklai, M

    1991-11-01

    After ingestion of 220 mg zinc sulfate, platelet aggregation was evaluated at various time intervals (i.e., T = 0, 1, and 3 hr) and the autologous plasma analyzed by atomic absorption analysis. The zinc levels increased maximally some 0.4 +/- 0.2 microgram/ml within 3 hr after ingestion, which for the entire blood pool corresponds to only 5% of the ingested zinc. Aggregation responses of platelet rich plasma (PRP), instigated with suboptimal levels of thrombin (less than 0.2 U/ml), ADP (less than 2 microM), epinephrine (less than 2 microM), collagen (less than 2 micrograms/ml), or PAF (less than 50 ng/ml), show significant improvement to at least one aggregant. Mean +/- SEM values for delta % aggregation increase are as follows: thrombin, 51 +/- 10%; epinephrine, 21 +/- 6%; ADP, 31 +/- 6%; collagen 23 +/- 6%; and platelet aggregating factor (PAF), 56 +/- 6%. For controls, the platelets from one individual with Glanzmann thrombasthenia as well as four undosed volunteers exhibited no significant changes in platelet responsiveness. Increased platelet responsiveness to agonists after zinc sulfate ingestion was observed in PRP from blood collected in either citrate or heparin. We demonstrate that within a relatively short time period, single bolus of nutritional zinc intake can significantly increase platelet reactivity. These findings show that nutritional zinc availability is relevant to hemostasis and may pertain to the viability of platelet concentrates in blood banks.

  18. Downregulation of Integrins in Cancer Cells and Anti-Platelet Properties Are Involved in Holothurian Glycosaminoglycan-Mediated Disruption of the Interaction of Cancer Cells and Platelets in Hematogenous Metastasis.

    PubMed

    Qian, Wenhui; Tao, Li; Wang, Yingyu; Zhang, Feng; Li, Mengqiu; Huang, Shile; Wang, Aiyun; Chen, Wenxing; Yue, Zhiqiang; Chen, Lei; Liu, Yuping; Huang, Chenhu; Zhang, Lei; Li, Yao; Lu, Yin

    2015-01-01

    Activated platelets have been recognized as an accessory character in the cascade of tumor hematogenous metastasis, and intervention of tumor cell attachment to the activated platelets or microemboli formation might be a leading strategy to prevent tumor cells surviving in the blood vessels and sequential metastasis. Recently, we have demonstrated that holothurian glycosaminoglycan (hGAG), a sulfated polysaccharide with potent anticoagulant activity extracted from the sea cucumber Holothuria leucospilota Brandt, was highly efficacious against tumor metastasis. In this study, we identified the potential effects of hGAG on the disruption of interactions of cancer cells and platelets and the underlying mechanisms, which were supported by the following evidence: hGAG (1) inhibited thrombin-induced platelet activation and aggregation, (2) reduced adhesion between platelet and breast cancer cells, and abrogated platelets/cancer cells adhering to fibrinogen, (3) attenuated platelet-cancer cell complex formation (the number and size of aggregates) and (4) suppressed both mRNA and protein levels of β1 and β3 integrins, matrix metalloproteinase (MMP)-2 and MMP-9, while increasing the expression of the MMP inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1 in MDA-MB-231 cells. These results suggested that both the antiplatelet properties and mitigation of the levels of cellular adhesion molecules contributed to the anticancer effects of hGAG, and might thus be exploited for clinical adjuvant therapy to attenuate tumor hematogenous metastasis.

  19. Evaluation of [3-(1-Methyl-1H–indol –3-yl-methylene)-2–oxo-2, 3–dihydro-1H- indole–5-sulfonamide] (OXSI-2), as a Syk selective inhibitor in platelets

    PubMed Central

    Bhavaraju, Kamala; Kim, Soochong; Daniel, James L.; Kunapuli, Satya P.

    2008-01-01

    In the present study, we characterized OXSI-2 ([3-(1-Methyl-1H–indol –3-yl-methylene)-2–oxo-2, 3–dihydro-1H- indole–5-sulfonamide], a putative inhibitor of Syk, and determined its specificity and selectivity in platelets. We found that OXSI-2 completely abolished convulxin-induced platelet functional responses. In order to determine whether OXSI-2 inhibited Src family kinase-mediated platelet responses, we evaluated its effect on Src family kinase (SFK)-mediated signaling events in platelets, viz. Lyn-mediated phosphorylation of Y352 on Syk, LAT- Y191 phosphorylation by Syk, and protease-activated receptor (PAR)-mediated phosphorylation of ERK. In the present work, we report that convulxin mediated Syk tyrosine 352 phosphorylation is not inhibited by OXSI-2, whereas piceatannol and PP2 abolished it. Syk-mediated Y191 LAT phosphorylation is abolished by all the three inhibitors. AYPGKF-induced phosphorylation of ERK was marginally inhibited by OXSI-2, whereas treatment with PP2 and piceatannol completely abolished it. However, PAR mediated thromboxane generation (an event mediated by ERK) was potentiated by OXSI-2 whereas PP2 and piceatannol brought thromboxane to basal levels. Protein kinase C (PKC) inhibitors are known to potentiate PAR-mediated thromboxane generation in platelets. In contrast, OXSI-2, unlike PKC inhibitors, did not inhibit secretion. Therefore, we conclude that OXSI-2 is not a Syk-selective inhibitor in platelets because of its unexplained non-specific effects. PMID:18068154

  20. 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

  1. 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.

  2. Natriuretic peptides induce weak VASP phosphorylation at Serine 239 in platelets.

    PubMed

    Borgognone, Alessandra; Lowe, Kate L; Watson, Stephen P; Madhani, Melanie

    2014-01-01

    Cyclic guanosine-3',5'-monophoshate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides (NP; e.g. atrial NP [ANP]), which activate soluble and particulate guanylyl cyclases, respectively. The role of NO in regulating cGMP and platelet function is well documented, whereas there is little evidence supporting a role for NPs in regulating platelet reactivity. By studying platelet aggregation and secretion in response to a PAR-1 peptide, collagen and ADP, and phosphorylation of the cGMP-dependent protein kinase (PKG) substrate vasodilator-stimulated phosphoprotein (VASP) at serine 239, we evaluated the effects of NPs in the absence or presence of the non-selective cGMP and cAMP phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Our results show that NPs, possibly through the clearance receptor (natriuretic peptide receptor-C) expressed on platelet membranes, increase VASP phosphorylation but only following PDE inhibition, indicating a small, localised cGMP synthesis. As platelet aggregation and secretion measured under the same conditions were not affected, we conclude that the magnitude of PKG activation achieved by NPs in platelets per se is not sufficient to exert functional inhibition of platelet involvement in haemostasis. PMID:23469931

  3. 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. PMID:26660525

  4. 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

  5. Platelet Disorders

    MedlinePlus

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

  6. Delineation of Platelet Activation Pathway of Scutellarein Revealed Its Intracellular Target as Protein Kinase C.

    PubMed

    Tian, Xiaoxuan; Chang, Lianying; Ma, Guangyin; Wang, Taiyi; Lv, Ming; Wang, Zhilong; Chen, Liping; Wang, Yuefei; Gao, Xiumei; Zhu, Yan

    2016-01-01

    Erigeron breviscapus has been widely used in traditional Chinese medicine (TCM) and its total flavonoid component is commonly used to treat ischemic stroke, coronary heart disease, diabetes and hypertension. Scutellarin is the major ingredient of E. breviscapus and scutellarein is one of the main bioactive metabolites of scutellarin in vivo, but the latter's pharmacological activities have not been fully characterized. Provided evidence that could inhibit platelet aggregation, the effect of scutellarein on rat washed platelets and its underlying mechanisms were evaluated in our research. Scutellarein inhibited platelet adhesion and aggregation induced by multiple G protein coupled receptor agonists such as thrombin, U46619 and ADP, in a concentration-dependent manner. Furthermore, the mild effect of scutellarein on intracellular Ca(2+) mobilization and cyclic AMP (cAMP) level was observed. On the other hand, the role of scutellarein as potential protein kinase C (PKC) inhibitor was confirmed by PKC activity analysis and molecular docking. The phorbol myristate acetate-induced platelets aggregation assay with or without ADP implied that the scutellarein takes PKC(s) as its primary target(s), and acts on it in a reversible way. Finally, scutellarein as a promising agent exhibited a high inhibition effect on ADP-induced platelet aggregation among its analogues. This study clarifies the PKC-related signaling pathway involved in antiplatelet action of scutellarein, and may be beneficial for the treatment of cardiovascular diseases. PMID:26581323

  7. Smoking further increases platelet activity in patients with mild hypertension.

    PubMed

    Gleerup, G; Winther, K

    1996-01-01

    In this study the authors examine whether smoking further heightens platelet activity and reduces fibrinolysis above that already present in mild hypertension. Ten smokers and 11 non-smokers, all with mild hypertension (defined as a diastolic pressure between 90 and 110 mm Hg) were compared for their platelet activity in vitro and in vivo and for their fibrinolytic activity. Successive measurements were made with the patients lying at rest after they had assumed the erect posture for 10 min and at the end of a 5-min moderately strenuous exercise test. The threshold for platelet aggregation by ADP in vitro was significantly lower in samples taken from the smokers at rest (1.4 +/- 0.9 mumol L(-1)) than in the non-smokers (3.5 +/- 2.5 mumol L(-1)), and the difference persisted both in the upright posture and after exercise. The level of platelet release of beta-thromboglobulin was, likewise, higher in the smokers in the upright posture. Neither standing up nor physical exercise had any significant influence on either of these two indices of platelet activity. The euglobulin clot lysis time was slightly longer in the smokers than in the non-smokers in all three experimental situations, but the differences were not significant. Inhibitor of tissue plasminogen activator was not materially different in the two groups (Table 2). The results indicate that smoking adds a further element of heightened platelet activity to that inherently present in hypertension.

  8. 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

    2016-10-01

    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. PMID:27406812

  9. Lactate is a possible mediator of the glucose effect on platelet inhibition.

    PubMed

    Kobzar, Gennadi; Mardla, Vilja; Samel, Nigulas

    2014-01-01

    Abstract Glucose has been found to impair the inhibition of platelets with aspirin and alter the basal activity of nitric oxide synthase (NOS) in platelets. The aim of this work was to study the effects of glucose on the inhibitory pathways in activated platelets. A short-term incubation of glucose impaired the inhibition of platelet aggregation induced by agents activating an NOS-dependent pathway, such as l-arginine, adenosine and α-tocopherol. However, glucose had no effect on the inhibition induced by iloprost and BW245C, agents that activate the cyclic adenosine monophosphate (cAMP) signaling pathway. Potassium lactate attenuated the effects of the same inhibitors as glucose did. The inhibitors of glucose transport prevented the effect of glucose. Dichloroacetate, known to prevent the conversion of pyruvate to lactate and to decrease lactate in platelets, significantly attenuated the effect of glucose in platelets. The data support the suggestion that the effect of glucose on the inhibition of platelets by agents activating an NOS-dependent pathway is mediated by glucose metabolite lactate. PMID:23909711

  10. 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).

  11. Platelets mediate acetaminophen hepatotoxicity.

    PubMed

    Lam, Fong W; Rumbaut, Rolando E

    2015-10-01

    In this issue of Blood, Miyakawa et al show that platelets and protease-activated receptor (PAR)-4 contribute to acetaminophen (APAP)-induced liver damage. Using various strategies in a mouse model of APAP overdose, the authors demonstrate that platelets participate in the progression of liver damage, and that the direct thrombin inhibitor lepirudin and PAR-4 deficiency attenuate hepatotoxicity. These findings have the potential to help identify future therapeutic targets for APAP-induced hepatotoxicity. PMID:26450954

  12. State of the Art in Platelet Function Testing

    PubMed Central

    E. Kehrel, Beate; F. Brodde, Martin

    2013-01-01

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

  13. Action of Caffeine as an Amyloid Inhibitor in the Aggregation of Aβ16-22 Peptides.

    PubMed

    Sharma, Bhanita; Paul, Sandip

    2016-09-01

    Alzheimer's disease (AD) is a neurodegenerative disease caused due to aggregation of Aβ peptides in the brain tissues. Recently, several studies on AD transgenic mice have shown the effect of caffeine in significantly reducing the Aβ amyloid level in their brains. However, the mechanism and mode of caffeine action on amyloid aggregation are not known. Therefore, in this study, we have carried out molecular dynamics simulations of five amyloid-forming Aβ16-22 peptides in pure water and in a regime of caffeine solutions, with different caffeine/peptide stoichiometric ratios. The secondary structure analyses of peptides in pure water show the formation of β-sheet conformations, whereas on addition of caffeine, these ordered conformations become negligible. The radial distribution function, contact map, nonbonding interaction energy, hydrogen bonding, potential of mean force, and hydration analyses show that there is less interpeptide interaction in the presence of caffeine, and the effect is greater with an increasing caffeine ratio. The interaction of aromatic phenylalanine residues of peptides with caffeine restricts the interpeptide interaction tendency. Upon increasing the number of caffeine molecules, interaction of caffeine with other hydrophobic residues also increases. Thus, the hydrophobic core-recognition motif of amyloid formation of peptides is physically blocked by caffeine, thereby abolishing the self-assembly formation. PMID:27487451

  14. Intracellular screening of a peptide library to derive a potent peptide inhibitor of α-synuclein aggregation.

    PubMed

    Cheruvara, Harish; Allen-Baume, Victoria L; Kad, Neil M; Mason, Jody M

    2015-03-20

    Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71-82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD.

  15. Intracellular Screening of a Peptide Library to Derive a Potent Peptide Inhibitor of α-Synuclein Aggregation*

    PubMed Central

    Cheruvara, Harish; Allen-Baume, Victoria L.; Kad, Neil M.; Mason, Jody M.

    2015-01-01

    Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson disease (PD). Studies have focused largely on residues 71–82, yet most early-onset mutations are located between residues 46 and 53. A semirationally designed 209,952-member library based entirely on this region was constructed, containing all wild-type residues and changes associated with early-onset PD. Intracellular cell survival screening and growth competition isolated a 10-residue peptide antagonist that potently inhibits α-syn aggregation and associated toxicity at a 1:1 stoichiometry. This was verified using continuous growth measurements and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity studies. Atomic force microscopy and circular dichroism on the same samples showed a random-coil structure and no oligomers. A new region of α-syn for inhibitor targeting has been highlighted, together with the approach of using a semirational design and intracellular screening. The peptides can then be used as candidates for modification in drugs capable of slowing or even preventing the onset of PD. PMID:25616660

  16. 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

  17. Impact of non-inhibited platelet supplementation on platelet reactivity in patients treated with prasugrel or ticagrelor for an acute coronary syndrome: An ex vivo study.

    PubMed

    Bonhomme, Fanny; Bonvini, Robert; Reny, Jean-Luc; Poncet, Antoine; Fontana, Pierre

    2015-01-01

    Managing bleeding in patients receiving P2Y12 inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in patients treated with prasugrel or ticagrelor. The aim of this study was to evaluate the minimal amount of platelet supplementation (in terms of ratio of non-inhibited platelets to inhibited platelets) necessary to restore platelet reactivity in platelet-rich plasma (PRP) of patients treated with aspirin and a prasugrel or ticagrelor loading dose for an acute coronary syndrome. PRP samples from patients were mixed ex vivo with increasing proportions of pooled PRP from healthy volunteers. Platelet reactivity was challenged with adenosine diphosphate (ADP), arachidonic acid, collagen or thrombin receptor activating peptide using light transmission aggregometry. The primary endpoint was the proportion of patient samples recovering an ADP-induced maximal aggregation (ADP-Aggmax) value above 40%. In patients treated with prasugrel (n = 32), ADP-Aggmax increased progressively with supplements of pooled PRP, with an average increase of 7.9% (95% CI [7.1; 8.8], p < 0.001) per each 20% increase in the ratio of non-inhibited platelets to inhibited platelets. A ratio of 60% was associated with 90% of patients reaching the primary endpoint. In patients treated with ticagrelor (n = 15), ADP-Aggmax did not significantly increase with any level of supplements. In conclusions, ex vivo addition of non-inhibited platelets significantly improved ADP-Aggmax in patients treated with prasugrel with a dose-dependent effect. There was no evidence of such a reversal in patients treated with ticagrelor. These results suggest that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor. PMID:25905916

  18. Gasotransmitters and platelets.

    PubMed

    Truss, Nicola J; Warner, Timothy D

    2011-11-01

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

  19. [Assessment of platelet function in man].

    PubMed

    Gaussem, Pascale

    2006-01-01

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

  20. Early intraplatelet signaling enhances the release of human platelet PAR-1 and -4 amino-terminal peptides in response to thrombin.

    PubMed

    Ofosu, Frederick A; Dewar, Lori; Song, Yingqi; Cedrone, Aisha C; Hortelano, Gonzalo; Craven, Sharon J

    2009-02-24

    Activation of washed human platelets initiated with alpha-thrombin, SFLLRN, or AYPGKF invariably results in the generation of PAR-1-(1-41) and PAR-4-(1-47). PAR-1-(1-41) and PAR-4-(1-47) are amino-terminal peptides generated when PAR-1 and -4 are cleaved in their first extracellular domains after R(41) and R(47), respectively, to expose the tethered ligand domains of PAR-1 and -4. Since soybean trypsin inhibitor decreases generation of PAR-1-(1-41) and PAR-4-(1-47) and other platelet aggregation-related responses to these three agonists, but does not inactivate alpha-thrombin, a platelet trypsin-like proteinase apparently activates PAR-1 and -4 to propagate PAR-dependent platelet responses. This study identified the signaling pathways implicated in the generation of the platelet proteinase that in turn produces PAR-1-(1-41) and PAR-4-(1-47), to thereby drive the subsequent PAR-dependent platelet aggregation-related responses to alpha-thrombin, SFLLRN, or AYPGKF. Only inhibitors of signaling enzymes that prevented ATP release (forskolin, PGE(1), or BIMI-1) prevented or delayed the generation of PAR-1-(1-41) and PAR-4-(1-47) in response to all three agonists. SBTI prevented platelet aggregation initiated by alpha-thrombin, SFLLRN, or AYPGKF but did so less effectively when it was added 10 s after each agonist. Thus, the platelet-derived proteinase acts within 10 s of each agonist addition to generate PAR-1-(1-41) and PAR-4-(1-47). Furthermore, alpha-thrombin may not effectively catalyze PAR-1-(1-41) and PAR-4-(1-47) generation. We propose that unidentified ATP-dependent phosphorylation reactions catalyzed by PKC help to generate the platelet-derived proteinase that propagates human platelet PAR-1 and -4 activation by the three agonists. PMID:19182900

  1. 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.

  2. Design, Synthesis and Structure-Activity Relationship (SAR) Studies of 2,4-Disubstituted Pyrimidine Derivatives: Dual Activity as Cholinesterase and Aβ-Aggregation Inhibitors

    PubMed Central

    Mohamed, Tarek; Zhao, Xiaobei; Habib, Lila K.; Yang, Jerry; Rao, Praveen P. N.

    2011-01-01

    A novel class of 2,4-disubstituted pyrimidines (7a–u, 8a–f, 9a–e) that possess substituents with varying steric and electronic properties at the C-2 and C-4 positions, were designed, synthesized and evaluated as dual cholinesterase and amyloid-β (Aβ)-aggregation inhibitors. In vitro screening identified N-(naphth-1-ylmethyl)-2-(pyrrolidin-1-yl)pyrimidin-4-amine (9a) as the most potent AChE inhibitor (IC50 = 5.5 μM). Among this class of compounds, 2-(4-methylpiperidin-1-yl)-N-(naphth-1-ylmethyl)pyrimidin-4-amine (9e) was identified as the most potent and selective BuChE inhibitor (IC50 = 2.2 μM, Selectivity Index = 11.7) and was about 5.7-fold more potent compared to the commercial, approved reference drug galanthamine (BuChE IC50 = 12.6 μM). In addition, the selective AChE inhibitor N-benzyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-amine (7d), exhibited good inhibition of hAChE-induced aggregation of Aβ1–40 fibrils (59% inhibition). Furthermore, molecular modeling studies indicate that a central pyrimidine ring serves as a suitable template to develop dual inhibitors of cholinesterase and AChE-induced Aβ aggregation thereby targeting multiple pathological routes in AD. PMID:21429752

  3. The role of platelets in inflammation.

    PubMed

    Thomas, Mark R; Storey, Robert F

    2015-08-31

    There is growing recognition of the critical role of platelets in inflammation and immune responses. Recent studies have indicated that antiplatelet medications may reduce mortality from infections and sepsis, which suggests possible clinical relevance of modifying platelet responses to inflammation. Platelets release numerous inflammatory mediators that have no known role in haemostasis. Many of these mediators modify leukocyte and endothelial responses to a range of different inflammatory stimuli. Additionally, platelets form aggregates with leukocytes and form bridges between leukocytes and endothelium, largely mediated by platelet P-selectin. Through their interactions with monocytes, neutrophils, lymphocytes and the endothelium, platelets are therefore important coordinators of inflammation and both innate and adaptive immune responses.

  4. Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

    PubMed

    Clemetson, Kenneth J

    2015-01-01

    Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.

  5. 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).

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

    PubMed Central

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

    2014-01-01

    Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders. PMID:24868545

  7. 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.

  8. 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

  9. 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

    2015-01-01

    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.

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

    PubMed

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

    2006-01-01

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

  11. Platelet-delivered therapeutics.

    PubMed

    Lyde, R; Sabatino, D; Sullivan, S K; Poncz, M

    2015-06-01

    We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

  12. 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

  13. Reaction of (/sup 3/H)-taurine maleimide with platelet surface thiols

    SciTech Connect

    Karl, D.W.; Mills, D.C.B.

    1986-05-01

    Taurine Maleimide (2-maleimidoethanesulfonate, TM) was synthesized from (2-/sup 3/H)-taurine and methoxycarbonylmaleimide (MCM). The yield of a 1 ..mu..mol synthesis approached 100% (based on taurine) when MCM was used in 4-fold excess. The product (TM*) was purified by ion exchange chromatography. TM* reacted irreversibly with thiol groups on the surface of washed human platelets, leading to incorporation of radioactivity into platelet pellets. Incorporation was blocked by cysteine, mercuribenzenesulfonate (MBS), dithiobisnitrobenzoate, and N-ethylmaleimide, but not by taurine or by inhibitors of anion transport. Reaction of TM* with platelets showed the dependence on time and concentration characteristics of a bimolecular reaction. The number of reactive sites ranged from 1 to 5 x 10/sup 5//platelet, and the apparent rate constant from 1 to 3 x 10/sup 3//(M x min). TM was less effective than MBS as an inhibitor of platelet aggregation induced by several agents. TM had no effect on the uptake of serotonin, taurine, or phosphate by the platelets, processes which are sensitive to MBS. These differences, considered with the similarity in size and charge of TM and MBS, suggest that classes of thiols defined as exofacial by their accessibility to MBS can differ substantially in their reactivity with other impermeant reagents.

  14. 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 d