Evaluation of Antiplatelet Activity of Phenolic Compounds by Flow Cytometry

Platelets play a pivotal role in coagulation, or clot formation, resulting in haemostasis, after endothelium injury. Disturbance of platelet activation may lead to pathologic thrombosis. Platelet activation and aggregation are common factors in atherothrombotic events, critical in the atherothrombotic process, and cardiovascular diseases. Several drugs are being used for antiplatelet therapy to prevent and/or treat atherosclerosis and cardiovascular diseases. Synthetic antiplatelet drugs hold possible undesired health consequences (cardiovascular diseases, carcinogenicity, etc.), advocating their replacement with natural, effective, and non-toxic compounds. Many phenolic compounds are created as secondary metabolites of plants, are found in many fruits and vegetables, and constitute a wide family of high-added-value molecules. Their biological activities include antioxidant, anti-platelet, and anti-inflammatory action. Based on the above, we examined five phenolic compounds (ellagic acid, ferulic acid, gallic acid, quercetin, and kaempferol) for their effect on platelet reactivity in whole blood samples using flow cytometry. Quantification of activated platelet marker CD62-P by flow cytometry showed that all five compounds inhibited platelet activation in vitro, induced by adenosine diphosphate (ADP) and collagen. Interestingly, based on the IC50 values obtained for expression of CD62-P, among ellagic, ferulic, and gallic acid, gallic acid showed significantly higher inhibition than the other two. Kaempferol found to be a more potent inhibitor than quercetin, following previously reported results from aggregometry. Results obtained from our flow cytometry screening indicate antiplatelet activity from novel phenolic compounds and their potential use as drugs for thrombosis and cardiovascular diseases.

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Abstract 28: Carboxyalkyl-pyrrole Phosphatidylethanolamine Adducts Are Present In Vivo And Induce Platelet Activation Via Toll-like Receptor

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a28 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Sudipta Biswas ◽

Soumya Panigrahi ◽

Alejandro Zimman ◽

Eugene Podrez

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Platelet Activation ◽

Platelet Reactivity ◽

Biological Activities ◽

Direct Interaction ◽

Covalent Modification ◽

Downstream Signaling

A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress. Lipid peroxidation, a major consequence of oxidative stress generates highly reactive products capable of modifying autologous proteins as well as lipids. Hydroxy-ω-oxoalkenoic acids and their carboxyalkylpyrrole (CAP) protein adducts are recently described products of lipid peroxidation with strong biological activity mediated by Toll like receptors (TLR). Phosphatidylethanolamine (PE) is the second most abundant phospholipid in the living organisms. While recent studies suggest that PE is a major target for covalent modification by reactive products of lipid peroxidation, the presence of such products in vivo, their biological activities and receptors involved are not established. We now report that CAP-PE adducts are present in vivo in circulation and are significantly elevated in plasma of hyperlipidemic apoE-/- mice. In vitro experiments demonstrated that CAP-PE adducts induce platelet integrin αIIbβ3 activation, P-selectin expression and promote platelet aggregation. Multiple complimentary approaches demonstrated that platelet activation by CAP-PE is mediated by TLR2 and TLR1. Furthermore, direct interaction of CAP-PE and TLR2 was demonstrated. CAP-PE induced assembly of TLR2/TLR1 receptor complex in platelets leading to downstream signaling via MyD88/TIRAP-dependent pathway. CAPs-PE induced signaling included phosphorylation and activation of IRAK4 and subsequent activation of TRAF6, Src family kinase, Syk and PLCγ2. Thus, our study identified carboxyalkylpyrrole adducts of phosphatidylethanolamine as novel end products accumulating in circulation in hyperlipidemia that can induce platelet activation via innate immunity signaling pathway.

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A Simple and Rapid Method to Determine GPIIb/IIIa-Receptor Inhibitor Activity in Whole Blood

Hämostaseologie ◽

10.1055/s-0038-1660401 ◽

1999 ◽

Vol 19 (03) ◽

pp. 134-138

Author(s):

Gitta Kühnel ◽

A. C. Matzdorff

Keyword(s):

Flow Cytometry ◽

Platelet Count ◽

Blood Sample ◽

Platelet Activation ◽

Whole Blood ◽

Receptor Occupancy ◽

Aggregate Formation ◽

Inhibitor Activity ◽

Receptor Inhibitor

SummaryWe studied the effect of GPIIb/IIIa-inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GPIIb/IIIa-inhibitors (c7E3, DMP728, XJ757), then thrombin or ADP were added and after 1 min the sample was fixed. Samples without c7E3 but with 0.1 U/ml thrombin had a decrease in platelet count. Samples with increasing concentrations of c7E3 had a lesser or no decrease in platelet count. The two other inhibitors (DMP 725, XJ757) gave similar results. GPIIb/IIIa-inhibitors prevent aggregate formation and more single platelets remain in the blood sample. The agonist-induced decrease in platelet count correlates closely with the concentration of the GPIIb/IIIa inhibitor and receptor occupancy. This correlation may be used as a simple measure for inhibitor activity in whole blood.

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The Effect of Heparin vs. Citrate on the Interaction of Platelets with Vascular Graft Materials

Thrombosis and Haemostasis ◽

10.1055/s-0038-1660145 ◽

1985 ◽

Vol 54 (04) ◽

pp. 842-848 ◽

Cited By ~ 14

Author(s):

Kandice Kottke-Marchant ◽

James M Anderson ◽

Albert Rabinovitch ◽

Richard A Huskey ◽

Roger Herzig

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Vascular Graft ◽

Time Course ◽

Platelet Reactivity ◽

Polymer Surfaces ◽

In Vitro Perfusion ◽

Citrate Anticoagulation ◽

Platelet Release

SummaryHeparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

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ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000494795 ◽

2018 ◽

Vol 50 (5) ◽

pp. 1779-1793 ◽

Cited By ~ 19

Author(s):

Xiang Wang ◽

Yun-Feng Fu ◽

Xiao Liu ◽

Guo Feng ◽

Dan Xiong ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Mtor Pathway ◽

Enhanced Chemiluminescence ◽

Transmission Electron ◽

Related Proteins

Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

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Isolation of hydroxytyrosol from olive leaves extract, radioiodination and investigation of bioaffinity using in vivo/in vitro methods

Radiochimica Acta ◽

10.1524/ract.2013.2068 ◽

2013 ◽

Vol 101 (9) ◽

pp. 585-593 ◽

Cited By ~ 1

Author(s):

M. Ozkan ◽

F. Z. Biber Muftuler ◽

A. Yurt Kilcar ◽

E. I. Medine ◽

P. Unak

Keyword(s):

Small Intestine ◽

Phenolic Compounds ◽

Biological Activities ◽

In Vivo Studies ◽

Flavonoid Content ◽

Olive Leaves ◽

In Vitro Methods ◽

Extraction And Purification

Summary It is known that medicinal plants like olive have biological activities due to their flavonoid content such as olueropein, tyrosol, hydroxytyrosol etc. In current study, hydroxytrosol (HT) which is one of the major phenolic compounds in olive, olive leaves and olive oil, was isolated after methanol extraction and purification of olive leaves which are grown in the northern Anatolia region of Turkey. The isolated HT was radiolabeled with 131I (131I-HT) and the bioaffinity of this radiolabeled component of olive leaves extract was investigated by using in vivo/in vitro methods. It was found that HT could be radiolabeled with 131I in yields of 95.6±4.4% (n = 8), and in vivo studies showed that 131I-HT is taken up by urinary bladder, stomach, small intestine, large intestine, breast and prostate. Significant incorporation of activity was observed in cell lines via in vitro studies.

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Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment

Blood ◽

10.1182/blood.v78.1.154.154 ◽

1991 ◽

Vol 78 (1) ◽

pp. 154-162 ◽

Cited By ~ 157

Author(s):

J Valles ◽

MT Santos ◽

J Aznar ◽

AJ Marcus ◽

V Martinez-Sales ◽

...

Keyword(s):

Platelet Activation ◽

Therapeutic Potential ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

Stimulus Response ◽

Absolute Requirement ◽

Thromboxane Production ◽

And Function ◽

Aspirin Ingestion

Abstract Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte- induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling.

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RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival

Blood ◽

10.1182/blood.2019003251 ◽

2020 ◽

Vol 136 (15) ◽

pp. 1773-1782 ◽

Cited By ~ 1

Author(s):

Daniel DeHelian ◽

Shuchi Gupta ◽

Jie Wu ◽

Chelsea Thorsheim ◽

Brian Estevez ◽

...

Keyword(s):

Platelet Count ◽

G Protein ◽

Platelet Activation ◽

Platelet Reactivity ◽

Rgs Proteins ◽

Platelet Recovery ◽

Platelet Production ◽

Platelet Survival

Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

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Use of d-erythro-sphingosine as a pharmacological inhibitor of protein kinase C in human platelets

Biochemical Journal ◽

10.1042/bj2780387 ◽

1991 ◽

Vol 278 (2) ◽

pp. 387-392 ◽

Cited By ~ 19

Author(s):

W A Khan ◽

S W Mascarella ◽

A H Lewin ◽

C D Wyrick ◽

F I Carroll ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Platelet Activation ◽

Biological Activities ◽

Dose Dependence ◽

Human Platelets ◽

Kinase C ◽

Similar Dose ◽

Induced Aggregation

Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.

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Anthocyanins from red cabbage extract — evidence of protective effects on blood platelets

Open Life Sciences ◽

10.2478/s11535-012-0057-9 ◽

2012 ◽

Vol 7 (4) ◽

pp. 655-663 ◽

Cited By ~ 7

Author(s):

Joanna Saluk ◽

Michał Bijak ◽

Joanna Kołodziejczyk-Czepas ◽

Małgorzata Posmyk ◽

Krystyna Janas ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Platelet Activation ◽

Blood Platelets ◽

Protective Effects ◽

Platelet Activity ◽

Red Cabbage ◽

Arachidonate Cascade ◽

Wide Range ◽

Antioxidative Effects

AbstractRed cabbage belongs to cruciferous vegetables recognized as a rich source of anthocyanins. Anthocyanins have a wide range of therapeutic advantages without adverse effects, including cardiovascular protective properties. For development of cardiovascular diseases, platelet activation is crucial; therefore compounds which inhibit platelet activation are sought after. The anti-platelet activity of anthocyanins has only been described and is still unclear. In our study, the extract of anthocyanins, obtained from fresh leaves of red cabbage, was used in vitro to examine their antioxidative effects on platelets under oxidative stress conditions which are responsible for hyperactivity of these cells. The antiplatelet and antioxidative activities were determined by platelet aggregation and specific markers of the arachidonate cascade with O2−· generation, and oxidative changes (carbonyl groups and 3-nitrotyrosine). Extracts (5–15 μM) protected platelet proteins and lipids against oxidative damage, and diminished platelet activation. Anthocyanins from red cabbage provided beneficial anti-platelet effects and might help prevent cardiovascular diseases.

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High-Added-Value Antioxidants Obtained from the Degradation of Wine Phenolics by Lactobacillus plantarum

Journal of Food Protection ◽

10.4315/0362-028x-70.11.2670 ◽

2007 ◽

Vol 70 (11) ◽

pp. 2670-2675 ◽

Cited By ~ 33

Author(s):

JOSÉ MARÍA LANDETE ◽

HÉCTOR RODRÍGUEZ ◽

BLANCA DE LAS RIVAS ◽

ROSARIO MUÑOZ

Keyword(s):

Phenolic Compounds ◽

Gallic Acid ◽

Lactobacillus Plantarum ◽

Antimicrobial Activities ◽

Added Value ◽

Methyl Gallate ◽

Biotechnological Production ◽

Wine Production ◽

High Concentrations ◽

Ferulic Acids

Disposal of the waste from wine production has long been a problem for wineries, mainly because of the presence of phenolic compounds. In this study, we analyzed the antimicrobial activities of 10 wine phenolic compounds against Lactobacillus plantarum strains. Inhibition increased in this order: catechin = gallic acid < epicatechin = salicylic acid < methyl gallate = caffeic acid < ferulic acid = tryptophol < p-coumaric acid. The obtained results indicated that L. plantarum is able to grow in the presence of high concentrations of some wine phenolic compounds. Of the 10 compounds analyzed, only the hydroxycinnamic acids, gallic acid, and methyl gallate were metabolized by the four L. plantarum strains studied. Results also revealed that 4-vinylphenol and 4-vinylguaiacol are originated from p-coumaric and ferulic acids. These phenolic compounds are valuable intermediates in the biotechnological production of new fragrances. In addition, gallic acid and its ester, methyl gallate, are metabolized to produce the powerful antioxidant pyrogallol. Therefore, it might be possible to use L. plantarum strains to obtain high-added-value antioxidants from the degradation of phenolic compounds found in wine wastes.

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