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

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Ye-Ming Lee

2012-01-01

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

The binding of fibrinogen to platelets in diabetes mellitus

International Nuclear Information System (INIS)

Minno, G. di; Cerbone, A.M.; Iride, C.; Mancini, M.

1986-01-01

Platelets from diabetics are known to be more sensitive in vitro to a variety of aggregating agents, to produce more prostaglandin endoperoxides and thromboxane and to bind more 125 I-fibrinogen than platelets from normal controls. Fibrinogen binding to platelets is a pre-requisite for platelet aggregation. Previous studies suggested a role for prostaglandins and/or thrombaxane A 2 in the exposure of fibrinogen receptors on platelets. The present study compares fibrinogen binding to hyperaggregable platelets from diabetic patients and to normal platelets when prostaglandin/thromboxane formation is suppressed by aspirin. It was found that pre-treatment with aspirin reduced collagen or thrombin-induced binding to platelets from none-retinopathic diabetics to the values seen in controls. By contrast, aspirin did not normalize binding to platelets obtained from retinopathic diabetics. The combination of aspirin with apyrase (an ADP scavenger) almost completely inhibited binding and aggregation of platelets from normal controls or non-retinophatic diabetics exposed to collagen or thrombin, whereas it only partially affected binding and aggregation of platelets from retinopathics. By using a monoclonal antibody (B59.2) to the platelet receptor for fibrinogen, we determined that this receptor was quantitatively and qualitatively the same on platelets from normal controls and diabetics. We conclude that increased fibrinogen binding and hyperaggregability of platelets from none-retinopathic diabetics is related to their capacity to form more prostaglandin endoperoxides/thromboxane than normal platelets. In contrast, hyperaggregability and increased binding of platelets from retinopathics appear at least partly related to a mechanism independent of ADP release and thromboxane synthesis. (Author)

Antiplatelet effect of phloroglucinol is related to inhibition of cyclooxygenase, reactive oxygen species, ERK/p38 signaling and thromboxane A2 production

International Nuclear Information System (INIS)

Chang, Mei-Chi; Chang, Hsiao-Hua; Chan, Chiu-Po; Chou, Han-Yi; Chang, Bei-En; Yeung, Sin-Yuet; Wang, Tong-Mei; Jeng, Jiiang-Huei

2012-01-01

Platelet dysfunction is a major risk factor of cardiovascular diseases such as atherosclerosis, stroke and myocardial infarction. Many antiplatelet agents are used for prevention and treatment of these diseases. In this study, phloroglucinol (2.5–25 μM) suppressed AA-induced platelet aggregation and thromboxane B 2 (TXB 2 ) production, but not U46619-induced platelet aggregation. Phloroglucinol (100–250 μM) showed little cytotoxicity to platelets. Phloroglucinol inhibited the COX-1 and COX-2 activities by 45–74% and 49–72% respectively at concentrations of 10–50 μM. At concentrations of 1 and 5 μM, phloroglucinol attenuated the AA-induced ROS production in platelets by 30% and 53%, with an IC 50 of 13.8 μM. Phloroglucinol also inhibited the PMA-stimulated ROS production in PMN. Preincubation of platelets by phloroglucinol (10–25 μM) markedly attenuated the AA-induced ERK and p38 phosphorylation. Intravenous administration of phloroglucinol (2.5 and 5 μmol/mouse) suppressed the ex vivo AA-induced platelet aggregation by 57–71%. Phloroglucinol administration also elevated the mice tail bleeding time. Moreover, phloroglucinol inhibited the IL-1β-induced PGE 2 production in pulp fibroblasts. These results indicate that antiplatelet and anti-inflammatory effects of phloroglucinol are related to inhibition of COX, ROS and TXA2 production as well as ERK/p38 phosphorylation in platelets. Phloroglucinol further suppress PMA-induced ROS production in PMN. The antiplatelet effect of phloroglucinol was confirmed by ex vivo study. Clinically, the consumption of phloroglucinol-containing food/natural products as nutritional supplement may be helpful to cardiovascular health. Phloroglucinol has potential pharmacological use. -- Highlights: ► Phloroglucinol suppressed AA-induced platelet aggregation and thromboxane production. ► Phloroglucinol inhibited COX activity and IL-1b-induced PGE2 production in fibroblast. ► Phloroglucinol declined platelet and

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

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Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

2017-01-15

Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Anti-thromboxane B2 antibodies protect against acetaminophen-induced liver injury in mice

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Ivan Ćavar

2011-12-01

Full Text Available Prostanoids are lipid compounds that mediate a variety of physiological and pathological functions in almost all body tissues and organs. Thromboxane (TX A2 is a powerful inducer of platelet aggregation and vasoconstriction and it has ulcerogenic activity in the gastrointestinal tract. Overdose or chronic use of a high dose of acetaminophen (N-acetyl-paminophenol, APAP is a major cause of acute liver failure in the Western world. We investigated whether TXA2 plays a role in host response to toxic effect of APAP. CBA/H Zg mice of both sexes were intoxicated with a single lethal or high sublethal dose of APAP, which was administered to animals by oral gavage. The toxicity of APAP was determined by observing the survival of mice during 48 h, by measuring concentration of alanine-aminotransferase (ALT in plasma 20-22 h after APAP administration and by liver histology. The results have shown that anti-thromboxane (TX B2 antibodies (anti-TXB2 and a selective inhibitor of thromboxane (TX synthase, benzylimidazole (BZI, were significantly hepatoprotective, while a selective thromboxane receptor (TPR antagonist, daltroban, was slightly protective in this model of acute liver injury. A stabile metabolite of TXA2, TXB2, and a stabile agonist of TPR, U-46619, had no influence on APAP-induced liver damage. Our findings suggest that TXA2 has a pathogenic role in acute liver toxicity induced with APAP, which was highly abrogated by administration of anti-TXB2. According to our results, this protection is mediated, at least in part, through decreased production of TXB2 by liver fragments ex vivo.

Cumulative inhibitory effect of low-dose aspirin on vascular prostacyclin and platelet thromboxane production in patients with atherosclerosis.

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Weksler, B B; Tack-Goldman, K; Subramanian, V A; Gay, W A

1985-02-01

The relationship between the antithrombotic and antiplatelet effects of aspirin is complex, since aspirin influences other systems that protect against thrombosis as well as inhibiting platelet function. We investigated possible cumulative effects of low-dose aspirin on vascular production of prostacyclin in patients with documented atherosclerotic cardiovascular disease. Candidates for coronary artery vein graft bypass ingested 20 mg of aspirin daily during the week before surgery, and platelet aggregation, platelet formation of thromboxane A2 (TXA2), aortic and saphenous vein production of prostacyclin (PGI2), and hemostatic status were measured at the time of the bypass surgery. Low-dose aspirin markedly inhibited platelet aggregation responses and reduced TXA2 generation by greater than 90%, effects similar to those observed with much higher doses of aspirin. Both aortic and saphenous vein production of PGI2 were inhibited by 50% compared with PGI2 produced by vascular tissues of control subjects who received no aspirin preoperatively (51 +/- 10 pg 6-keto-PGF1 alpha/mg aortic wet weight [mean +/- SEM] in aspirin-treated subjects vs 130 +/- 16 pg/mg in control subjects, and 71 +/- 8 pg/mg saphenous vein wet weight vs 131 +/- 17 pg/mg). Blood loss at surgery was not significantly increased by preoperative low-dose aspirin as measured by chest tube drainage (754 +/- 229 ml in aspirin-treated subjects vs 645 +/- 271 ml in control subjects), hematocrit nadir (31.2 +/- 1.9% vs 31.8 +/- 1.7%), or transfusions (2.2 +/- 1.3 units of red blood cells vs 2.2 +/- 1.7 units).(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity column for purification of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor

International Nuclear Information System (INIS)

Venton, D.L.; Arora, S.K.; Kim, S.O.; Lim, C.T.; Le Breton, G.C.

1987-01-01

The TXA 2 /PGH 2 receptor antagonist, 13-azaprostanoic acid (13-APA), was synthesized and used as the immobilized ligand in the affinity column purification of the 13-APA/U46619 binding component in human platelets. Diazo coupling of the ligand to the phenol of this tyr-gly-gly-NH-(CO)-O-Sepharose gave the affinity column material. Isolated platelet membranes were solubilized with detergent, applied directly to the affinity column and the eluate collected as 6 x 70 ml fractions. For each fraction, protein concentration and specific 3 H-13-APA/numberH-U46619 binding were determined. The majority of the applied protein (>98%) eluted in fraction number1. However, the specific 13-APA/U46619 binding per mg of protein was localized in fractions number4 and number5, representing approximately a 500-fold purification of this binding component. These results suggest that the platelet TXA 2 /PGH 2 receptor protein is retarded by this column, and that starting from crude, solubilized platelet membranes, a single pass through the column provides a 500-fold purification of the receptor

Evidence that the angiotensin at 2-receptor agonist compound 21 is also a low affinity thromboxane TXA2-receptor antagonist

DEFF Research Database (Denmark)

Fredgart, M.; Leurgans, T.; Stenelo, M.

2015-01-01

Objective: The objective of this study was to test whether Compound 21 (C21), a high-affinity, non-peptide angiotensinAT2-receptor agonist, is also an antagonist of thromboxane A2 (TXA2) receptors thus reducing both vasoconstriction and platelet aggregation. Design and method: Binding of C21...... to the TXA2 receptor was determined by TBXA2R Arrestin Biosensor Assay. Mouse mesenteric arteries were mounted in wire myographs, and responses to increasing concentrations of C21 (1nM- 10muM) were recorded during submaximal contractions with 0.1muM U46619 (TXA2 analogue) or 1muMphenylephrine. To control for......AT2-receptor specificity, arteries were pre-incubated with the AT2-receptor antagonist PD123319 (10muM), or mesenteric arteries from AT2-receptor knock-out (AT2R-/y) mice were used. An inhibitory effect of C21 (100nM - 10muM) on U46619 (0,3muM) induced platelet aggregation was examined in whole human...

Characterization of the human gene (TBXAS1) encoding thromboxane synthase.

Science.gov (United States)

Miyata, A; Yokoyama, C; Ihara, H; Bandoh, S; Takeda, O; Takahashi, E; Tanabe, T

1994-09-01

The gene encoding human thromboxane synthase (TBXAS1) was isolated from a human EMBL3 genomic library using human platelet thromboxane synthase cDNA as a probe. Nucleotide sequencing revealed that the human thromboxane synthase gene spans more than 75 kb and consists of 13 exons and 12 introns, of which the splice donor and acceptor sites conform to the GT/AG rule. The exon-intron boundaries of the thromboxane synthase gene were similar to those of the human cytochrome P450 nifedipine oxidase gene (CYP3A4) except for introns 9 and 10, although the primary sequences of these enzymes exhibited 35.8% identity each other. The 1.2-kb of the 5'-flanking region sequence contained potential binding sites for several transcription factors (AP-1, AP-2, GATA-1, CCAAT box, xenobiotic-response element, PEA-3, LF-A1, myb, basic transcription element and cAMP-response element). Primer-extension analysis indicated the multiple transcription-start sites, and the major start site was identified as an adenine residue located 142 bases upstream of the translation-initiation site. However, neither a typical TATA box nor a typical CAAT box is found within the 100-b upstream of the translation-initiation site. Southern-blot analysis revealed the presence of one copy of the thromboxane synthase gene per haploid genome. Furthermore, a fluorescence in situ hybridization study revealed that the human gene for thromboxane synthase is localized to band q33-q34 of the long arm of chromosome 7. A tissue-distribution study demonstrated that thromboxane synthase mRNA is widely expressed in human tissues and is particularly abundant in peripheral blood leukocyte, spleen, lung and liver. The low but significant levels of mRNA were observed in kidney, placenta and thymus.

Roxicam pharmacological modulation of the prostacyclin-thromboxane system in heart failure-complicated acute myocardial infarction

International Nuclear Information System (INIS)

Shushlyapin, O.I.; Shelest, A.N.; Khossejn Shakhavat, A.F.M.

1991-01-01

The prostaglandin-thromboxane system, platelet hemostasis and central hemodynamics were evaluated in 51 patients with heart failure-complicated acute myocardial infarction. The concentration of active metabolites of thromboxane-prostacyclin system was determined by means of radioimmunoassay. The new non-steroidal antiinflammatory agent roxicam was shown to selectively inhibit thromboxane, without affecting prostacyclin levels. The agent may be use in therapy in patients with myocardial infarction concurrent with heart failure

A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

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Mumford, Andrew D; Dawood, Ban B; Daly, Martina E; Murden, Sherina L; Williams, Michael D; Protty, Majd B; Spalton, Jennifer C; Wheatley, Mark; Mundell, Stuart J; Watson, Steve P

2010-01-14

We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.

Kinetics of prostaglandin E2 and thromboxane A2 synthesis and suppression of PHA-stimulated peripheral blood mononuclear leucocytes.

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Awara, W; Hillier, K; Jones, D

1986-12-01

The immunomodulatory effects of thromboxane A2 and prostaglandin E2 on peripheral blood mononuclear leucocytes stimulated with PHA in vitro, and the relationship of this to the time-course of their synthesis in culture, were investigated using prostaglandin E2, a thromboxane A2 synthesis inhibitor (UK37248), a thromboxane A2 mimic (U46619) and a thromboxane A2 receptor blocker (EP045). The inhibitory effect of prostaglandin E2 on PHA-induced human peripheral blood mononuclear leucocyte proliferation diminishes if the addition of PGE2 is delayed. If added 4 hr after a maximum concentration of PHA (5 micrograms/ml), the effect of PGE2 was reduced by 60%. If a submaximal concentration of PHA (1 microgram/ml) was used, the effect of PGE2 was not reduced if added 4 hr later but fell by about 60% after 16 hr. UK37248 moderately inhibited PHA-induced activation while substantially inhibiting thromboxane A2 synthesis and simultaneously enhancing PGE2 synthesis. The enhanced accumulation of PGE2 occurs while sensitivity to PGE2 is dropping. U46619, exogenously applied as a thromboxane A2 mimic, inhibited PHA-induced activation at concentrations that did not significantly alter PGE2 synthesis. EP045, which may modulate the effects of endogenous thromboxane A2 by blocking receptors, did not alter PHA-induced activation. We conclude that thromboxane A2 may have a role in inhibiting PHA-induced activation on the basis of the effect of U46619. However, this study highlights difficulties in utilizing prostaglandin and thromboxane receptor and synthesis inhibitors to examine their endogenous role in the modulation of mitogen-induced activation in vitro. If sensitivity to the purported endogenous substance is limited to the early stages of culture and if only low levels are synthesized at this early stage, then blocking drugs would have little effect.

Reduced methylation of the thromboxane synthase gene is correlated with its increased vascular expression in preeclampsia.

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Mousa, Ahmad A; Strauss, Jerome F; Walsh, Scott W

2012-06-01

Preeclampsia is characterized by increased thromboxane and decreased prostacyclin levels, which predate symptoms, and can explain some of the clinical manifestations of preeclampsia, including hypertension and thrombosis. In this study, we examined DNA methylation of the promoter region of the thromboxane synthase gene (TBXAS1) and the expression of thromboxane synthase in systemic blood vessels of normal pregnant and preeclamptic women. Thromboxane synthase is responsible for the synthesis of thromboxane A(2), a potent vasoconstrictor and activator of platelets. We also examined the effect of experimentally induced DNA hypomethylation on the expression of thromboxane synthase in a neutrophil-like cell line (HL-60 cells) and in cultured vascular smooth muscle and endothelial cells. We found that DNA methylation of the TBXAS1 promoter was decreased and thromboxane synthase expression was increased in omental arteries of preeclamptic women as compared with normal pregnant women. Increased thromboxane synthase expression was observed in vascular smooth muscles cells, endothelial cells, and infiltrating neutrophils. Experimentally induced DNA hypomethylation only increased expression of thromboxane synthase in the neutrophil-like cell line, whereas tumor necrosis factor-α, a neutrophil product, increased its expression in cultured vascular smooth muscle cells. Our study suggests that epigenetic mechanisms and release of tumor necrosis factor-α by infiltrating neutrophils could contribute to the increased expression of thromboxane synthase in maternal systemic blood vessels, contributing to the hypertension and coagulation abnormalities associated with preeclampsia.

Serum thromboxane B2 (TXB2 Determination is Influenced by Sample Incubation Temperature in Healthy Beagle Dogs

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Aleš Jerin

2009-01-01

Full Text Available The measurement of serum thromboxane B2 (TXB2 production by platelets is a specific test for assessment of platelet cyclooxygenase (COX-1 activity following administration of non-steroidal anti-inflammatory drugs (NSAIDs. The aim of this study was to investigate the influence of sample incubation at 37 °C for one hour on serum TXB2 concentration in comparison with incubation at room temperature. A total of 54 blood samples for serum TXB2 measurements were collected from six healthy beagle dogs into two separate serum tubes. While one group of tubes was incubated in a 37 °C water bath, the second group of tubes was left to coagulate at room temperature, both for one hour. Serum TXB2 concentrations were measured by ELISA. The mean concentration (± SD of serum TXB2 in the group of samples that were incubated at 37 °C was significantly (P 2 concentration in healthy beagle dogs and demonstrate that validated methods for assessment of COX-1 activity by measurement of serum TXB2 should be used in order to make results more reliable and comparable between different studies. The results of this study might be of great help in planning NSAID studies in dogs by providing the information that TXB2 generation by platelets is influenced profoundly by incubation temperature.

Urinary Thromboxane B2 and Thromboxane Receptors in Bladder Cancer: Opportunity for Detection and Monitoring

OpenAIRE

Moussa, Omar; Ciupek, Andrew; Watson, Dennis K.; Halushka, Perry V.

2011-01-01

We have previously found increased expression of thromboxane synthase (TXAS) and thromboxane receptor (TP) beta isoform in the tissues of patients with bladder cancer. Studies in cell lines and mice have indicated a potential significant role of the thromboxane signaling pathway in the pathogenesis of human bladder cancer. This study was designed to determine if the changes observed in the tissues of patients with bladder cancer were mirrored by changes in the urine of these patients. We foun...

Influence of whole-body gamma irradiation upon arachidonic acid metabolism in rat platelets

International Nuclear Information System (INIS)

Lognonne, J.L.; Ducousso, R.; Rocquet, G.; Kergonou, J.F.

1985-01-01

The effects of whole-body gamma irradiation (8.4 Gy) were studied on arachidonic acid (AA) metabolism in rat's blood platelets, from day D + 1 to day D + 10 after irradiation. AA conversion into thromboxane B 2 (TxB 2 ) increased at D + 1 and then gradually decreased to very low values from D + 7 to D + 10. This decrease in the conversion of exogenous AA into TxB 2 was due to a lower AA incorporation into platelets and not to a decrease of cyclooxygenase and thromboxane-synthetase activities. AA incorporation into membrane phospholipids of blood platelets was much more decreased than AA incorporation into whole platelets; moreover, the lipid composition of the platelet membranes was markedly modified after irradiation, which must have resulted in structural and functional changes in these membranes; from these effects of whole-body gamma irradiation on platelets, the latter's membranes appeared as a major site of in vivo radiation damage in these cells

The purification step is not crucial in EIA measurements of thromboxane B2 and 11-dehydrothromboxane B2 in human plasma.

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Sadilkova, Lenka; Paluch, Zoltan; Mottlova, Jirina; Bednar, Frantisek; Alusik, Stefan

2012-01-01

Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes. For the purpose of this study, 20 plasma samples (10 healthy donors, 10 patients under treatment with acetylsalicylic acid) were screened for TxB2 and 11-dTxB2 concentrations using commercial competitive EIA kits (Cayman Chemicals, Tallinn, Estonia; Neogen, Lexington, KY, USA) with or without the introduction of the purification procedure. The purification step does not significantly affect the results of EIA measurements of the two of TxA2 metabolites (TxB2, 11-dTxB2) in human plasma. The levels of TxB2 and 11-dTxB2 determined in the plasma samples were not significantly changed (p < 0.05) when the purification step was omitted compared to the purified samples. This study establishes a protocol allowing for reliable and reproducible plasma TxB2 and 11-dTxB2 EIA measurement for routine basic screening of platelet function.

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation

OpenAIRE

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

2013-01-01

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-depende...

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

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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. Copyright © 2015 John Wiley & Sons, Ltd.

Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

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Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

2010-01-01

Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

Prostaglandin E2 and thromboxane B2 release from human monocytes treated with bacterial lipopolysaccharide

International Nuclear Information System (INIS)

Nichols, F.C.; Garrison, S.W.; Davis, H.W.

1988-01-01

We investigated the capacity of counterflow-isolated human monocytes to independently synthesize thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) when stimulated with bacterial lipopolysaccharide (LPS). Independent metabolism was confirmed by establishing different specific activities (dpm/ng) of TxB2 and PGE2 released from LPS-treated cells. For metabolites released during the initial 2-hr treatment period, the specific activity of PGE2 was approximately threefold higher than that of TxB2 regardless of labeling with [3H]arachidonic acid (AA) or [14C]AA. Cells that were pulse-labeled for 2 hr with [3H]AA demonstrated a decreasing PGE2 specific activity over 24 hr, whereas the TxB2 specific activity remained unchanged. In contrast, cells continuously exposed to [14C]AA demonstrated an increasing TxB2 specific activity that approached the level of PGE2 by 24 hr. These results suggest the presence of at least 2 cyclooxygenase metabolic compartments in counterflow-isolated monocytes. Although freshly isolated monocytes have been reported to contain variable numbers of adherent platelets, additional experiments demonstrated that counterflow-isolated platelets are not capable of releasing elevated levels of TxB2 or PGE2 when treated with LPS. It is proposed from these findings that at least two subsets of monocytes exist in peripheral blood that can be distinguished on the basis of independent conversion of AA to TxB2 and PGE2

Meal-induced platelet activation in diabetes mellitus type 1 or type 2 is related to postprandial insulin rather than glucose levels.

Science.gov (United States)

Spectre, Galia; Stålesen, Ragnhild; Östenson, Claes-Göran; Hjemdahl, Paul

2016-05-01

Postprandial platelet activation was related to postprandial insulin rather than glucose levels in a previous meal insulin study in type 2 diabetes mellitus (T2DM). We therefore compared postprandial platelet activation in type 1 (T1DM) patients without insulin secretion and T2DM patients with high postprandial insulin levels. Patients with T1DM (n=11) and T2DM (n=12) were studied before and 90min after a standardized meal without premeal insulin. Five T1DM patients volunteered for a restudy with their regular premeal insulin. Platelet activation was assessed by flow cytometry, with and without the thromboxane analogue U46619 or ADP, and by whole blood aggregometry (Multiplate®). Effects of insulin (100μU/mL) in vitro were also studied. Before the meal, glucose, insulin and platelet activation markers other than platelet-leukocyte aggregates (PLAs) were similar in T1DM and T2DM; PLAs were higher in T1DM. Postprandial glucose levels increased more markedly in T1DM (to 22.1±1.4 vs. 11.2±0.6mmol/L) while insulin levels increased only in T2DM (from 24.4±4.4 to 68.8±12.3μU/mL). Platelet P-selectin expression, fibrinogen binding and PLA formation stimulated by U46619 were markedly enhanced (approximately doubled) and whole blood aggregation stimulated by U46619 was increased (pinsulin in T1DM patients showed postprandial platelet activation when postprandial insulin levels increased. In vitro insulin mildly activated platelets in both groups. Postprandial platelet activation via the thromboxane pathway is related to postprandial hyperinsulinemia and not to postprandial hyperglycaemia in patients with diabetes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of the TEG® platelet mappingTM assay in blood donors

DEFF Research Database (Denmark)

Bochsen, Louise; Wiinberg, Bo; Kjelgaard-Hansen, Mads Jens

2007-01-01

for quantification of platelet function, including the contribution of the adenosine diphosphate (ADP) and thromboxane A2 (TxA2) receptors to clot formation. Methods In 43 healthy blood donors, the analytical (CVa) and inter-individual variability (CVg) of the TEG® Platelet MappingTM assay were determined together......Background Monitoring of antiplatelet therapy in patients at cardiovascular risk is difficult because existing platelet function tests are too sophisticated for clinical routine. The whole blood TEG® Platelet MappingTM assay measures clot strength as maximal amplitude (MA) and enables...

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

Science.gov (United States)

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.

Beta-lactam antibiotic-induced platelet dysfunction: Evidence for irreversible inhibition of platelet activation in vitro and in vivo after prolonged exposure to penicillin

International Nuclear Information System (INIS)

Burroughs, S.F.; Johnson, G.J.

1990-01-01

beta-Lactam antibiotics cause platelet dysfunction with bleeding complications. Previous in vitro studies documented reversible inhibition of agonist-receptor interaction. This mechanism is inadequate to explain the effect of beta-lactam antibiotics in vivo. Platelet function does not return to normal immediately after drug treatment, implying irreversible inhibition of platelet function. We report here evidence of irreversible platelet functional and biochemical abnormalities after in vitro and in vivo exposure to beta-lactam antibiotics. Irreversible binding of [14C]-penicillin (Pen) occurred in vitro. After 24 hours' in vitro incubation with 10 to 20 mmol/L Pen, or ex vivo after antibiotic treatment, irreversible functional impairment occurred; but no irreversible inhibition of alpha 2 adrenergic receptors, measured with [3H]-yohimbine, or high-affinity thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, measured with agonist [3H]-U46619 and antagonist [3H]-SQ29548, occurred. However, low-affinity platelet TXA2/PGH2 receptors were decreased 40% after Pen exposure in vitro or in vivo, indicating irreversible membrane alteration. Two postreceptor biochemical events were irreversibly inhibited in platelets incubated with Pen for 24 hours in vitro or ex vivo after antibiotic treatment. Thromboxane synthesis was inhibited 28.3% to 81.7%. Agonist-induced rises in cytosolic calcium ([Ca2+]i) were inhibited 40.1% to 67.5% in vitro and 26.6% to 52.2% ex vivo. Therefore, Pen binds to platelets after prolonged exposure, resulting in irreversible dysfunction attributable to inhibition of TXA2 synthesis and impairment of the rise in [Ca2+]i. The loss of low-affinity TXA2/PGH2 receptors suggests that the primary site of action of these drugs is on the platelet membrane

DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

International Nuclear Information System (INIS)

Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

2010-01-01

Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 ± 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% ± 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 ± 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

The Effect of Disinfection on Viability and Function of Baboon Red Blood Cells and Platelets

Science.gov (United States)

1997-07-11

blood cells was evaluated by their ability to transport oxygen as assessed by measurement of 2,3 diphosphoglycerate (DPG)14 and red blood cell p50,15...Blood collected from the bleeding time site (referred to as "shed blood") had a significantly reduced thromboxane A2 level . The ability of the...preserved or treated platelets to increase the shed blood thromboxane A2 level and reduce the 8; extended bleeding time is the measure of their

Effect of acetyl salicylic acid on increased production of thromboxane after aortic graft surgery.

Science.gov (United States)

Lewin, J; Swedenborg, J; Egberg, N; Vesterqvist, O; Green, K

1989-06-01

Contact between blood and foreign surfaces, e.g. vascular grafts, causes activation and release of platelets. One consequence of platelet activation is production of thromboxane A2 (TxA2). The physiological effects of TxA2, i.e. platelet aggregation and vaso-constriction are counteracted by another prostanoid, prostacyclin (PGI2). Acetylsalicylic acid (ASA) causes a longlasting inhibition of platelet TxA2 production and a more shortlasting inhibition of PGI2 production. The present study examines TxA2 and PGI2 synthesis in patients receiving synthetic arterial grafts, some of which were treated with ASA. The prostanoid synthesis was evaluated by measurement of their main urinary metabolites with gas chromatography-mass spectrometry. Platelet release was evaluated by measurements of beta-thromboglobulin (beta-TG) and the plasma coagulation by measurements of fibrinopeptide A (FPA). These compounds were also measured in urine in order to avoid artifacts caused by activation of platelets and plasma coagulation during blood sampling. Following replacement of the abdominal aorta with a synthetic vascular graft there was a marked increase in the synthesis of TxA2 and PGI2. Increased levels of beta-TG and FPA were also demonstrated. Administration of ASA on the first and second postoperative days significantly reduced the synthesis of TxA2 but caused no significant effects on the other parameters measured. It is concluded that ASA may be beneficial in the postoperative period since it counteracts TxA2 with vasoconstricting and platelet aggregating properties but leaves PGI2 with vasodilating and antiaggregating properties relatively uneffected.

Aspirin decreases platelet uptake on Dacron vascular grafts in baboons

International Nuclear Information System (INIS)

Mackey, W.C.; Connolly, R.J.; Callow, A.D.

1984-01-01

The influence of a single dose of aspirin (5.4-7.4 mg/kg) on platelet uptake on 4-mm Dacron interposition grafts was studied in a baboon model using gamma camera scanning for 111-Indium labeled platelets. In vitro assessment of platelet function after aspirin administration revealed that in the baboon, as in the human, aspirin abolished arachidonic acid-induced platelet aggregation, prolonged the lag time between exposure to collagen and aggregation, and decreased plasma thromboxane B2 levels. Aspirin also prolonged the template bleeding time. Scans for 111-Indium labeled platelets revealed that pretreatment with a single dose of aspirin decreased platelet uptake on 4-mm Dacron carotid interposition grafts. This decrease in platelet uptake was associated with a significant improvement in 2-hour graft patency and with a trend toward improved 2-week patency

Platelet apoptosis by cld-induced glycoportein Ibα clustering

DEFF Research Database (Denmark)

van der Wal, Dianne E; Du, V X; Lo, KS

2010-01-01

Summary Background:  Cold-storage of platelets followed by rewarming induces changes in Glycoprotein (GP) Ibα-distribution indicative of receptor clustering and initiates thromboxane A2-formation. GPIbα is associated with 14-3-3 proteins, which contribute to GPIbα-signaling and in nucleated cells...

Platelet thromboxane B2-formation in end-stage kidney disease and after kidney transplantation

International Nuclear Information System (INIS)

Stefanovic, V.; Lecic, N.

1986-01-01

The aim of this work was to analyse TxB 2 formation by platelets in endstage kidney disease patients and in kidney graft recipients. Four groups of patients were studied: 12 preterminal chronic renal failure patients, 42 patients on maintenance hemodialysis, 8 patients on CAPD and 11 grafted patients. TxB 2 production by platelets was determined in serum following spontaneous blood clotting for 1/2 h at 37 0 C. Hemodialysis patients generated 80.7 ± 9.6 ng/ml (mean ± S.E.M.) of TxB 2 which was significantly (p 2 formation in hemodialysis patients had no relationship with the residual kidney function. Patients on CAPD produced 65.0 ± 12.7 ng/ml of TxB 2 . Very low TxB 2 generation was obtained also in preterminal chronic renal failure patients (57.0 ± 11.8 ng/ml). Kidney graft recipients had a mean TxB 2 production of 81.6 ± 24.2 ng/ml with a range from 12.5-200 ng/ml. Very low TxB 2 was formed in grafted patients with renal failure. (orig.) [de

Radioimmunological determination of thromboxane release in cardiac anaphylaxis

Energy Technology Data Exchange (ETDEWEB)

Anhut, H; Bernauer, W; Peskar, B A [Freiburg Univ. (Germany, F.R.). Dept. of Pharmacology

1977-07-01

A sensitive and specific radioimmunoassay for thromboxane B/sub 2/ was developed. Using the radioimmunoassay, substantial amounts of thromboxane B/sub 2/ were detected in perfusates of anaphylactic guinea pig hearts. Indomethacin decreased thromboxane B/sub 2/ levels in the perfusates to below the detection limit of the radioimmunoassay and concomitantly delayed the onset of coronary vasoconstriction after antigenic challenge.

Thromboxane A2 increases endothelial permeability through upregulation of interleukin-8

International Nuclear Information System (INIS)

Kim, Su-Ryun; Bae, Soo-Kyung; Park, Hyun-Joo; Kim, Mi-Kyoung; Kim, Koanhoi; Park, Shi-Young; Jang, Hye-Ock; Yun, Il; Kim, Yung-Jin; Yoo, Mi-Ae; Bae, Moon-Kyoung

2010-01-01

Thromboxane A 2 (TXA 2 ), a major prostanoid formed from prostaglandin H 2 by thromboxane synthase, is involved in the pathogenesis of a variety of vascular diseases. In this study, we report that TXA 2 mimetic U46619 significantly increases the endothelial permeability both in vitro and in vivo. U46619 enhanced the expression and secretion of interleukin-8 (IL-8), a major inducer of vascular permeability, in endothelial cells. Promoter analysis showed that the U46619-induced expression of IL-8 was mainly regulated by nuclear factor-κB (NF-κB). U46619 induced the activation of NF-κB through IκB kinase (IKK) activation, IκB phosphorylation and NF-κB nuclear translocation. Furthermore, the inhibition of IL-8 or blockade of the IL-8 receptor attenuated the U46619-induced endothelial cell permeability by modulating the cell-cell junctions. Overall, these results suggest that U46619 promotes vascular permeability through the production of IL-8 via NF-κB activation in endothelial cells.

Radioimmunoassay for platelet activation specific protein GMP-140 on the platelet surface and in plasma

International Nuclear Information System (INIS)

Wu Guoxin; Li Jianyong; Ruan Changgeng

1991-08-01

Using monoclonal antibody (McAb) SZ-51 which is specific for an alpha-granule membrane protein (GMP-140) on the surface of human activated platelets, the platelet GMP-140 expression in fixed whole blood was measured by direct radioimmunoassay and GMP-140 microparticles in plasma was measured by sandwich method. The GMP-140 molecules per platelet or milliliter (mL) were calculated for the following subjects; acute myocardial infarction; cerebro thrombosis; diabetic mellitus; asthma attack; epidemic hemorrhagic fever etc.. By comparing with the concentration of thromboxane B 2 (TXB 2 ) and von Willebrand factor (vWF) in plasma, it is confirmed that the measurement of GMP-140 molecules is better than that of TXB 2 and vWF. It is a sensitive and specific method for evaluating the platelet activation degree in vivo. The establishment of this method will be useful to diagnosing the thrombotic disorders and studying the pathogenesis of some other diseases

Niacin and biosynthesis of PGD2 by platelet COX-1 in mice and humans

Science.gov (United States)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A.; Wilensky, Robert L.; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A.

2012-01-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1–dependent formation of PGD2 and PGE2 followed by COX-2–dependent production of PGE2. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD2 receptor DP1. NSAID-mediated suppression of COX-2–derived PGI2 has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD2. Here, we show that PGD2 biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1–derived PGD2 biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD2 was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD2, like PGI2, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy. PMID:22406532

Hypersensitivity to thrombin of platelets from hypercholesterolemic rats

International Nuclear Information System (INIS)

Winocour, P.D.; Rand, M.L.; Kinlough-Rathbone, R.L.; Mustard, J.F.

1986-01-01

Hypersensitivity of platelets to thrombin has been associated with hypercholesterolemia. The authors have examined the mechanisms involved in this hypersensitivity. Rats were given diets rich in milk fat and containing added cholesterol and taurocholate to produce hypercholesterolemia (HC) (262 +/- 25 mg%) or added sitosterol as a normocholesterolemic control (NC) (89 +/- 6 mg%). Washed platelets were prelabelled with 14 C-serotonin. In the presence of acetylsalicyclic acid (ASA) (to inhibit thromboxane A 2 (TXA 2 ) formation) and creatine phosphate/creatine phosphokinase (CP/CPK) (to remove released ADP), HC platelets aggregated more (26 +/- 1%) and released more 14 C (9.1 +/- 2.0%) than NC platelets (aggregation: 0%, p 14 C release: 1.5 +/- 0.5%, p 2 formation is involved in the hypersensitivity of HC platelets to thrombin. Total binding of 125 I-thrombin to HC platelets was less than that to NC platelets but HC platelets were smaller and had less protein than NC platelets; the thrombin binding per mg platelet protein was the same for HC and NC platelets, indicating that hypersensitivity to thrombin of HC platelets does not result from increased thrombin binding. Thus, hypersensitivity of HC platelets to thrombin is not due to TXA 2 formation, the action of released ADP or increased thrombin binding

Effect of racemic ibuprofen dose on the magnitude and duration of platelet cyclo-oxygenase inhibition: relationship between inhibition of thromboxane production and the plasma unbound concentration of S(+)-ibuprofen.

Science.gov (United States)

Evans, A M; Nation, R L; Sansom, L N; Bochner, F; Somogyi, A A

1991-02-01

1. Four healthy male subjects received racemic ibuprofen (200, 400, 800 and 1200 mg), orally, on four occasions, 2 weeks apart, according to a four-way Latin-square design, in order to investigate the influence of increasing dose of ibuprofen on the magnitude and duration of its antiplatelet effect as well as on the relationship between such effect and drug concentration. 2. The antiplatelet effect of ibuprofen was assessed by measuring the inhibition of platelet thromboxane B2 (TXB2) generation during the controlled clotting of whole blood. The plasma unbound concentration of S(+)-ibuprofen, the enantiomer shown in an in vitro study to be responsible for the inhibitory effect of platelet TXB2 generation, was measured using an enantioselective method. 3. The maximum percentage inhibition of TXB2 generation increased significantly with dose from a mean +/- s.d. of 93.4 +/- 1.2% after the 200 mg dose to 98.8 +/- 0.3% after the 1200 mg dose, and there was an increase with dose in the duration of inhibition of TXB2 generation. The effect of ibuprofen on platelet TXB2 generation was transient and mirrored the time-course of unbound S(+)-ibuprofen in plasma; on all but one of the 16 occasions, serum TXB2 concentrations returned to at least within 10% of the pretreatment concentrations within 24 h of ibuprofen administration. 4. For each subject, the relationship between the percentage inhibition of TXB2 generation and the unbound concentration of S(+)-ibuprofen in plasma was modelled according to a sigmoidal Emax equation. The mean plasma unbound concentration of S(+)-ibuprofen required to inhibit platelet TXB2 generation by 50% (EC50) was 9.8 +/- 1.0 micrograms l-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

Science.gov (United States)

Eriksson, Andreas C; Jonasson, Lena; Lindahl, Tomas L; Hedbäck, Bo; Whiss, Per A

2009-01-01

Background Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA) and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. Methods With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2)-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. Results The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP)-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49). In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN-induced activation measured by flow

Partial purification and identification of the thrombozane A2/prostaglandin H2 receptor protein in human platelets

International Nuclear Information System (INIS)

Lim, C.T.; Kattelman, E.J.; Arora, S.K.; Venton, D.L.; Le Breton, G.C.

1986-01-01

The thromboxane A 2 /prostaglandin H 2 (TXA 2 /PGH 2 ) receptor antagonist [ 3 H]-13-azaprostanoic acid (13-APA) was used to identify and purify the platelet TXA 2 /PGH 2 receptor protein. Optimal solubilization of the 13-APA binding protein was achieved by extraction with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS) detergent. Preliminary purification of the crude solubilized membrane fraction was performed by gel filtration chromatography using a Sepharose 4B column. Further purification was accomplished by high performance liquid chromatography (HPLC) using a Synchropak GPC-500 column. The HPLC protein profile revealed two protein peaks, only one of which was enriched in [ 3 H]-13-APA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of this peak revealed two bands with molecular weights of 65,000 and 60,000 daltons. In binding studies using the 60,000 dalton-enriched subfraction, unlabelled 13-APA, the TXA 2 /PGH 2 mimetic U46619 and the TXA 2 /PGH 2 antagonist SQ 29,548 all competed for [ 3 H]-13-APA binding whereas TXB 2 did not compete for binding. Heat denaturation of this subfraction resulted in a complete loss of binding activity. These findings indicate that a protein of approximately 60,000 daltons represents the human platelet TXA 2 /PGH 2 receptor

Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis.

Science.gov (United States)

Lombard, Stephanie E; Pollitt, Alice Y; Hughes, Craig E; Di, Ying; Mckinnon, Tom; O'callaghan, Chris A; Watson, Steve P

2017-11-01

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A 2 (TxA 2 ). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.

The measurement of platelet activation by radioimmunoassay in asthma

International Nuclear Information System (INIS)

Wu Guoxin; Sun Jian; Li Jianyong; Ruan Changgeng

1992-02-01

Radioimmunoassay with specific monoclonal antibody was used to evaluate the platelet activation in 14 cases of acute bronchial asthma. The result showed that the number of molecules of alpha-granule membrane protein (GMP-140) which was exposed on the surface of platelet following secretion significantly increased on the surface of platelet and in plasma, while the number of molecules of glycoprotein (GP) I b and GPIII a did not change significantly; the concentration of thromboxane B 2 in plasma was raised, while the concentration of 6-keto-PGF 1a was within the normal limits; the concentrations of β-thromboglobulin (β-TG) and platelet factor 4(PF 4 ) in plasma increased significantly; the number of platelets decreased. These results strongly confirmed that the degree of platelet activation was enhanced during acute asthmatic attack. The significance of platelet activation in the pathogenesis of asthma should be further investigated

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y12 receptor.

Science.gov (United States)

Cunningham, Margaret R; Aungraheeta, Riyaad; Mundell, Stuart J

2017-07-05

Genetic variations in G protein-coupled receptor (GPCR) genes can disrupt receptor function in a wide variety of human genetic diseases, including platelet bleeding disorders. Platelets are critical for haemostasis with inappropriate platelet activation leading to the development of arterial thrombosis, which can result in heart attack and stroke whilst decreased platelet activity is associated with an increased risk of bleeding. GPCRs expressed on the surface of platelets play key roles in regulating platelet activity and therefore function. Receptors include purinergic receptors (P2Y 1 and P2Y 12 ), proteinase-activated receptor (PAR1 and PAR4) and thromboxane receptors (TPα), among others. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis. With the advance of genomic technologies, there has been a substantial increase in the identification of naturally occurring rare and common GPCR variants. These variants include single-nucleotide polymorphisms (SNPs) and insertion or deletions that have the potential to alter GPCR expression or function. A number of defects in platelet GPCRs that disrupt receptor function have now been characterized in patients with mild bleeding disorders. This review will focus on rare, function-disrupting variants of platelet GPCRs with particular emphasis upon mutations in the P2Y 12 receptor gene that affect receptor traffic to modulate platelet function. Further this review will outline how the identification and characterization of function-disrupting GPCR mutations provides an essential link in translating our detailed understanding of receptor traffic and function in cell line studies into relevant human biological systems. Copyright © 2017. Published by Elsevier B.V.

Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

Directory of Open Access Journals (Sweden)

Hedbäck Bo

2009-06-01

Full Text Available Abstract Background Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. Methods With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. Results The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49. In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN

Lung, aorta, and platelet metabolism of 14C-arachidonic acid in vitamin E deficient rats

International Nuclear Information System (INIS)

Valentovic, M.A.; Gairola, C.; Lubawy, W.C.

1982-01-01

14 C-arachidonic acid metabolism was determined in aortas, platelets, and perfused lungs from rats pair fed a basal diet supplemented with 0 or 100 ppm vitamin E for 11 weeks. Spontaneous erythrocyte hemolysis tests showed 92% and 8% hemolysis for the 0 and 100 ppm vitamin E groups, respectively. Elevated lung homogenate levels of malonaldehyde in the 0 ppm group confirmed its deficient vitamin E status. Aortas from the vitamin E deficient group synthesized 54% less prostacyclin than aortas from the supplemented group (p less than 0.05). Although thromboxane generation by platelets from the vitamin E deficient group exhibited a 37% increase, this difference was not statistically significant compared to the supplemented animals. Greater amounts of PGE2, PGF2 alpha, TXB2, and 6-keto-PGF1 alpha were obtained in albumin buffer perfusates from lungs of vitamin E deficient rats than in those from supplemented rats. Significant differences (p less than 0.05) were noticed, however, only for PGE2 and PGF2 alpha. These studies indicate that vitamin E quantitatively alters arachidonic acid metabolism in aortic and lung tissue but its effect on thromboxane synthesis by platelets is less marked

Rare platelet GPCR variants: what can we learn?

Science.gov (United States)

Nisar, S P; Jones, M L; Cunningham, M R; Mumford, A D; Mundell, S J

2015-07-01

Platelet-expressed GPCRs are critical regulators of platelet function. Pharmacological blockade of these receptors forms a powerful therapeutic tool in the treatment and prevention of arterial thrombosis associated with coronary atherosclerosis and ischaemic stroke. However, anti-thrombotic drug therapy is associated with high inter-patient variability in therapeutic response and adverse bleeding side effects. In order to optimize the use of existing anti-platelet drugs and to develop new therapies, more detailed knowledge is required relating to the molecular mechanisms that regulate GPCR and therefore platelet function. One approach has been to identify rare, function-disrupting mutations within key platelet proteins in patients with bleeding disorders. In this review, we describe how an integrated functional genomics strategy has contributed important structure-function information about platelet GPCRs with specific emphasis upon purinergic and thromboxane A2 receptors. We also discuss the potential implications these findings have for pharmacotherapy and for understanding the molecular basis of mild bleeding disorders. © 2014 The British Pharmacological Society.

Mechanisms Involved in Thromboxane A2-induced Vasoconstriction of Rat Intracavernous Small Penile Arteries

DEFF Research Database (Denmark)

Grann, Martin; Comerma Steffensen, Simon Gabriel; Arcanjo, Daniel Dias Rufino

2015-01-01

relaxation in rat mesenteric arteries. Our findings suggest that U46619 contraction depends on Ca2+ influx through L-type and TRP channels, and ROCKdependent mechanisms in penile arteries. Inhibition of the ROCK pathway is a potential approach for the treatment of erectile dysfunction associated......Diabetes is associated with erectile dysfunction and with hypercontractility in erectile tissue and this is in part ascribed to increased formation of thromboxane. Rho kinase (ROCK) is a key regulator of calcium sensitization and contraction in vascular smooth muscle. This study investigated...... the role of calcium and ROCK in contraction evoked by activation of the thromboxane receptors. Rat intracavernous penile arteries were mounted for isometric tension and intracellular calcium ([Ca2+]i) recording and corpus cavernosum for measurements of MYPT1 phosphorylation. In penile arteries, U46619...

Effects of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1991-02-01

Human platelet-rich plasma was irradiated with various doses (0,1.25,2.5,5.0,10,20,40Gy) of 60 Co γ-ray so as to observing the changes of metabolites and releasing substances of platelets. Alpha-granule membrane protein (GMP-140) molecules on the surface of platelet was expressed significantly increasing when the dosage of 60 Co γ-ray was 5 Gy; however, the glycoprotein (GP) I b and III a was not changed significantly; thromboxane B 2 production in plasma was significantly elevated while the γ-ray was only 2.5 Gy; the concentration of von Willebrand factor was increased when the γ-ray was over 5 Gy, this is in accordance with the GMP-140 molecules. These results indicate that platelets could be activated in vitro when the dosage of 60 Co γ was over 5 Gy

Platelet cyclooxygenase expression in normal dogs.

Science.gov (United States)

Thomason, J; Lunsford, K; Mullins, K; Stokes, J; Pinchuk, L; Wills, R; McLaughlin, R; Langston, C; Pruett, S; Mackin, A

2011-01-01

Human platelets express both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). Variation in COX-2 expression could be a mechanism for variable response to aspirin. The hypotheses were that circulating canine platelets express COX-1 and COX-2, and that aspirin alters COX expression. The objective was to identify changes in platelet COX expression and in platelet function caused by aspirin administration to dogs. Eight female, intact hounds. A single population, repeated measures design was used to evaluate platelet COX-1 and COX-2 expression by flow cytometry before and after aspirin (10 mg/kg Q12h for 10 days). Platelet function was analyzed via PFA-100(®) (collagen/epinephrine), and urine 11-dehydro-thromboxane B(2) (11-dTXB(2)) was measured and normalized to urinary creatinine. Differences in COX expression, PFA-100(®) closure times, and urine 11-dTXB(2 ): creatinine ratio were analyzed before and after aspirin administration. Both COX-1 and COX-2 were expressed in canine platelets. COX-1 mean fluorescent intensity (MFI) increased in all dogs, by 250% (range 63-476%), while COX-2 expression did not change significantly (P = 0.124) after aspirin exposure, with large interindividual variation. PFA-100(®) closure times were prolonged and urine 11-dTXB(2) concentration decreased in all dogs after aspirin administration. Canine platelets express both COX isoforms. After aspirin exposure, COX-1 expression increased despite impairment of platelet function, while COX-2 expression varied markedly among dogs. Variability in platelet COX-2 expression should be explored as a potential mechanism for, or marker of, variable aspirin responsiveness. Copyright © 2011 by the American College of Veterinary Internal Medicine.

Effects of carprofen, meloxicam and deracoxib on platelet function in dogs.

Science.gov (United States)

Mullins, Kathleen B; Thomason, John M; Lunsford, Kari V; Pinchuk, Lesya M; Langston, Vernon C; Wills, Robert W; McLaughlin, Ronald M; Mackin, Andrew J

2012-03-01

To determine effects of anti-inflammatory doses of COX-2 selective NSAIDs carprofen, meloxicam, and deracoxib on platelet function in dogs and urine 11-dehydro-thromboxane B2. Randomized, blocked, crossover design with a 14-day washout period. Healthy intact female Walker Hounds aged 1-6 years and weighing 20.5-24.2 kg. Dogs were given NSAIDs for 7 days at recommended doses: carprofen (2.2 mg kg(-1), PO, every 12 hours), carprofen (4.4 mg kg(-1), PO, every 24 hours), meloxicam (0.2 mg kg(-1), PO, on the 1st day then 0.1 mg kg(-1), PO, every 24 hours), and deracoxib (2 mg kg(-1), PO, every 24 hours). Collagen/epinephrine and collagen/ADP PFA-100 cartridges were used to evaluate platelet function before and during and every other day after administration of each drug. Urine 11-dehydro-thromboxane B(2) was also measured before and during administration of each drug. All NSAIDs significantly prolonged PFA-100 closure times when measured with collagen/epinephrine cartridges, but not with collagen/ADP cartridges. The average duration from drug cessation until return of closure times (collagen/epinephrine cartridges) to baseline values was 11.6, 10.6, 11 and 10.6 days for carprofen (2.2 mg kg(-1) every 12 hours), carprofen (4.4 mg kg(-1) every 24 hours), meloxicam and deracoxib, respectively. Oral administration of some COX-2 selective NSAIDs causes detectable alterations in platelet function in dogs. As in humans, PFA-100 collagen/ADP cartridges do not reliably detect COX-mediated platelet dysfunction in dogs. Individual assessment of platelet function is advised when administering these drugs prior to surgery, particularly in the presence of other risk factors for bleeding. © 2011 The Authors. Veterinary Anaesthesia and Analgesia. © 2011 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists.

The role of Odontella aurita, a marine diatom rich in EPA, as a dietary supplement in dyslipidemia, platelet function and oxidative stress in high-fat fed rats

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Haimeur Adil

2012-10-01

Full Text Available Abstract Background Dietary changes are a major factor in determining cardiovascular risk. n-3 polyunsaturated fatty acids modulate the risk factors for metabolic syndrome via multiple mechanisms, including the regulation of the lipid metabolism. We therefore investigated the effect of Odontella aurita, a microalga rich in EPA, which is already used as a food supplement, on the risk factors for high-fat diet induced metabolic syndrome in rats. Methods Male Wistar rats were divided into 4 groups and were fed with a standard diet (control; with the standard diet supplemented with 3% freeze-dried O. aurita (COA; with a high-fat diet (HF; or with the high-fat diet supplemented with 3% of freeze-dried O. aurita (HFOA for 7 weeks. In this study we evaluated the impact of these different diets on the risk factors for metabolic syndrome, such as hyperlipidemia, platelet aggregation, thromboxane B2 production, and oxidative stress. Results After 7 weeks of treatment, high fat feeding had increased final body weight, glycemia, triacylglycerol, and total cholesterol levels in plasma and liver compared to the control diet. Collagen-induced platelet aggregation and basal platelet thromboxane B2 were also higher in the high-fat fed rats than in those in the control group. In the liver, oxidative stress was greater in the HF group than in the control group. O. aurita intake in HFOA-fed rats resulted in lower glycemia and lipid levels in the plasma and liver relative than in the HF group. Thus, in the HFOA group, n-3 polyunsaturated fatty acid levels in the tissues studied (plasma, liver, and platelets were higher than in the HF group. Platelet hyper-aggregability tended to decrease in HFOA-fed rats as basal platelet thromboxane B2 production decreased. Finally, O. aurita reduced oxidative stress in the liver, with lower malondialdehyde levels and increased glutathione peroxidase activity. Conclusions O. aurita is a marine diatom rich in EPA as well as in other

The role of Odontella aurita, a marine diatom rich in EPA, as a dietary supplement in dyslipidemia, platelet function and oxidative stress in high-fat fed rats.

Science.gov (United States)

Haimeur, Adil; Ulmann, Lionel; Mimouni, Virginie; Guéno, Frédérique; Pineau-Vincent, Fabienne; Meskini, Nadia; Tremblin, Gérard

2012-10-31

Dietary changes are a major factor in determining cardiovascular risk. n-3 polyunsaturated fatty acids modulate the risk factors for metabolic syndrome via multiple mechanisms, including the regulation of the lipid metabolism. We therefore investigated the effect of Odontella aurita, a microalga rich in EPA, which is already used as a food supplement, on the risk factors for high-fat diet induced metabolic syndrome in rats. Male Wistar rats were divided into 4 groups and were fed with a standard diet (control); with the standard diet supplemented with 3% freeze-dried O. aurita (COA); with a high-fat diet (HF); or with the high-fat diet supplemented with 3% of freeze-dried O. aurita (HFOA) for 7 weeks. In this study we evaluated the impact of these different diets on the risk factors for metabolic syndrome, such as hyperlipidemia, platelet aggregation, thromboxane B2 production, and oxidative stress. After 7 weeks of treatment, high fat feeding had increased final body weight, glycemia, triacylglycerol, and total cholesterol levels in plasma and liver compared to the control diet. Collagen-induced platelet aggregation and basal platelet thromboxane B2 were also higher in the high-fat fed rats than in those in the control group. In the liver, oxidative stress was greater in the HF group than in the control group. O. aurita intake in HFOA-fed rats resulted in lower glycemia and lipid levels in the plasma and liver relative than in the HF group. Thus, in the HFOA group, n-3 polyunsaturated fatty acid levels in the tissues studied (plasma, liver, and platelets) were higher than in the HF group. Platelet hyper-aggregability tended to decrease in HFOA-fed rats as basal platelet thromboxane B2 production decreased. Finally, O. aurita reduced oxidative stress in the liver, with lower malondialdehyde levels and increased glutathione peroxidase activity. O. aurita is a marine diatom rich in EPA as well as in other bioactive molecules, such as pigments. The synergistic effect

Effect of 60Co γ-ray irradiation on human platelets

International Nuclear Information System (INIS)

Wu Guoxin; Zhao Yiming; Ruan Changgeng

1992-01-01

Human platelet-rich plasma was irradiated with various doses (0, 1.25, 2.5, 5, 10, 20, 40 Gy) of 60 Co γ-rays in order to observe the changes of metabolites and the release of internal substance of platelets. At the dose of 5 Gy, alpha-granule membrane protein (GMP-140) molecules expressed on the surface of platelets increased significantly, while glycoproteins (GP) Ib and IIIa did not change apparently; at the dose as low as of 2.5 Gy, thromboxane B 2 production in plasma was remarkably increased; and, at the dose of over 5 Gy, the concentration of von Willebrand factor increased with increasing doses as in the case of GMP-140 molecules. These results indicate that platelets can be activated in vitro when the dose of 60 Co γ-rays exceeds 5 Gy

Subarachnoid hemorrhage induces enhanced expression of thromboxane A2 receptors in rat cerebral arteries

DEFF Research Database (Denmark)

Ansar, Saema; Larsen, Carl; Maddahi, Aida

2010-01-01

Cerebral ischemia remains the key cause of morbidity and mortality after subarachnoid hemorrhage (SAH) with a pathogenesis that is still poorly understood. The aim of the present study was to examine the involvement of thromboxane A(2) receptors (TP) in the pathophysiology of cerebral ischemia...

Effects of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, on platelet activity.

Science.gov (United States)

Lee, Ki-Seon; Khil, Lee-Yong; Chae, Sang-Ho; Kim, Deukjoon; Lee, Byung-Hoon; Hwang, Gwi-Seo; Moon, Chang-Hyun; Chang, Tong-Shin; Moon, Chang-Kiu

2006-02-02

In the present study, the mechanism of antiplatelet activity of DK-002, a synthesized (6aS,cis)-9,10-Dimethoxy-7,11b-dihydro-indeno[2,1-c]chromene-3,6a-diol, was investigated. DK-002 inhibited the thrombin, collagen, and ADP-induced rat platelet aggregation in a concentration-dependent manner, with IC50 values of 120, 27, and 47 microM, respectively. DK-002 also inhibited thrombin-induced dense granule secretion, thromboxane A2 synthesis, and [Ca2+]i elevation in platelets. DK-002 did not show any significant effect on ADP-induced inhibition of cyclic AMP elevation by prostaglandin E1, but DK-002 was confirmed to inhibit ADP-induced [Ca2+]i elevation and shape change. DK-002 inhibited 4-bromo-A23187-induced [Ca2+]i elevation in the presence of creatine phosphate/creatine phosphokinase (CP/CPK, a ADP scavenging system) and indomethacin (a specific inhibitor of cyclooxygenase). DK-002 also inhibited Ca2+ mobilization in thrombin- or 4-bromo-A23187-stimulated platelets through its inhibitory effects on both Ca2+ release from intracellular stores and Ca2+ influx, in the presence of CP/CPK and indomethacin. Taken together, the present study shows that DK-002 has inhibitory effects on stimulation of platelets, and suggests that its antiplatelet activity might be related to the inhibitory mechanism on Ca2+ mobilization in stimulated platelets.

Evidence that platelet buoyant density, but not size, correlates with platelet age in man

International Nuclear Information System (INIS)

Mezzano, D.; Hwang, K.; Catalano, P.; Aster, R.H.

1981-01-01

Following infusion of 51Cr-labeled autologous platelets into normal subjects, high-density (HD) and low-density (LD) platelet cohorts were isolated by prolonged centrifugation in isosmotic arabino-galactan (Stractan). Specific radio-activity of LD platelets declined rapidly post-infusion (T1/2 . 1.5 days), but specific radioactivity of HD platelets remained constant or increased over a 3--4-day period and gradually declined for 6--7 days thereafter. These differences were exaggerated when platelet cohorts enriched in LD or HD cells by slow centrifugation in high-density albumin were labeled and transfused. Mean survival of a platelet cohort enriched with HD cells was significantly (P less than 0.02) shorter (7.73 days) than that of a cohort enriched with LD cells (9.33) days). In normal subjects treated with aspirin, capacity for thromboxane synthesis was regained more rapidly (P less than 0.05) in LD than in HD platelets. HD and LD platelets differed only slightly in mean volume (HD platelets . 7.57 mu3, LD platelets . 6.87 mu3, 0.05 less than P less than 0.01). We believe the most logical interpretation of these findings is that under normal conditions in man, newly formed platelets are less dense on the average than total platelets and become more dense as they age in the circulation. Thus, specific radioactivity of LD platelets declines rapidly as these platelets move into a more dense compartment and are replaced by newly formed, unlabelled cells; specific radioactivity of HD platelets remains constant or increases as labelled platelets enter this compartment in numbers equal to or greater than the number leaving it at the end of their life span. The similarity in mean volumes of LD and HD platelets suggests that platelet size is unrelated to platelet age under normal conditions

Radiation-induced changes in production of prostaglandins Fsub(2α), E, and thromboxane B2 in guinea pig parenchymal lung tissues

International Nuclear Information System (INIS)

Steel, L.K.; Catravas, G.N.

1982-01-01

At 1 hour to 4 days after unilateral exposure of guinea pigs to a single dose (0.5, 1.5, or 3.0 Gy) of gamma-radiation, changes were detected in prostaglandin and thromboxane concentrations in parenchymal lung tissues. At 1-3 hours after exposure, tissue levels of PGFsub(2α), PGE, and thromboxane B 2 were significantly elevated in animals receiving 3.0 Gy, with the magnitude of alteration revealing a radiation dose effect. By 24 hours, tissue prostaglandin and thromboxane levels returned to near control values. Lung tissue synthesis of prostaglandins in response to H-1 receptor stimulation by the exogenous addition of histamine revealed similar radiation dose effects. The carboxylic acid ionophore A23187, exogenously applied to lung tissues, revealed a transient peak of increased sensitivity to ionophore stimulation for TxB 2 synthesis at 24 hours and for PGFsub(2α) at 72 hours post-irradiation. The data suggest that significant alterations in prostaglandin and thromboxane concentrations in parenchymal lung tissues occur following irradiation, in a dose-dependent manner, and that altered responsiveness to H-1 receptor stimulation and divalent cation transport also occur

Supplementation with mixed tocopherols increases serum and blood cell gamma-tocopherol but does not alter biomarkers of platelet activation in subjects with type 2 diabetes.

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Clarke, Michael W; Ward, Natalie C; Wu, Jason H Y; Hodgson, Jonathan M; Puddey, Ian B; Croft, Kevin D

2006-01-01

Some studies have shown potential benefit of vitamin E on platelet function, but several clinical trials failed to show improved cardiovascular outcome with alpha-tocopherol supplementation. Gamma-tocopherol, a major dietary form of vitamin E, may have protective properties different from those of alpha-tocopherol. We compared the effects of supplementation with alpha-tocopherol (500 mg) and a gamma-tocopherol-rich compound (500 mg, containing 60% gamma-tocopherol) on serum and cellular tocopherol concentrations, urinary tocopherol metabolite excretion, and in vivo platelet activation in subjects with type 2 diabetes. Fifty-eight subjects were randomly assigned to receive either 500 mg alpha-tocopherol/d, 500 mg mixed tocopherols/d, or matching placebo. Serum, erythrocyte, and platelet tocopherol and urinary metabolite concentrations were measured at baseline and after the 6-wk intervention. Soluble CD40 ligand, urinary 11-dehydro-thromboxane B2, serum thromboxane B2, soluble P-selectin, and von Willebrand factor were measured as biomarkers of in vivo platelet activation. Serum alpha-tocopherol increased with both tocopherol treatments. Serum and cellular gamma-tocopherol increased 4-fold (P tocopherol group, whereas red blood cell gamma-tocopherol decreased significantly after alpha-tocopherol supplementation. Excretion of alpha-carboxyethyl-hydroxychroman increased significantly after supplementation with alpha-tocopherol and mixed tocopherols. Excretion of gamma-carboxyethyl-hydroxychroman increased significantly after supplementation with mixed tocopherols and after that with alpha-tocopherol, which may reflect the displacement of gamma-tocopherol by alpha-tocopherol due to incorporation of the latter into lipoproteins in the liver. Neither treatment had any significant effect on markers of platelet activation. Supplementation with alpha-tocopherol decreased red blood cell gamma-tocopherol, whereas mixed tocopherols increased both serum alpha-tocopherol and

Thromboelastography platelet mapping in healthy dogs using 1 analyzer versus 2 analyzers.

Science.gov (United States)

Blois, Shauna L; Banerjee, Amrita; Wood, R Darren; Park, Fiona M

2013-07-01

The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer's guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.

Niacin and biosynthesis of PGD₂ by platelet COX-1 in mice and humans

DEFF Research Database (Denmark)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela

2012-01-01

during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis....... Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased...... thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular...

Radioiodsodestannylation. Convenient synthesis of a high affinity thromboxane A2/prostaglandin H2 receptor antagonist

International Nuclear Information System (INIS)

Mais, D.E.; Hamanaka, Nobuyuki

1991-01-01

Radioiodination of methyl-7-[(2R, 2S, 5R)-6,6-dimethyl-3-(4-trimethylstannylbenzenesulfononylamino3S) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoate with [ 125 I] Na using a modification of the chloramine-T method in organic solvent is simple with high yields and site specific. The product, following hydrolysis of the ester, 7-[(2R, 2S, 3S, 5R)-6,6-dimethyl-3-(4[ 125 I]-iodobenzenesulfonylamino) bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid [( 125 I]-ISAP), was purified by HPLC. The high specific activity and specific binding will make the ligand a useful tool for the characterization of thromboxane A 2 /prostaglandin H 2 receptors. (author)

Cigarette smoke exposure alters [14C]arachidonic acid metabolism in aortas and platelets of rats fed various levels of selenium and vitamin E

International Nuclear Information System (INIS)

Valentovic, M.A.; Gairola, C.; Lubawy, W.C.

1985-01-01

Rats were placed on a basal diet supplemented with 0, 0.03, or 3 ppm selenium and 0 or 20 ppm vitamin E for 41-43 wk. Selenium deficiency decreased hepatic glutathione peroxidase activity and lowered both aortic prostacyclin (PGI2) and platelet thromboxane (TXA2) production compared to selenium- and vitamin E-supplemented animals. Vitamin E deficiency increased hepatic lipid peroxidation and decreased aortic PGI2 synthesis. Rats exposed daily for 31-32 wk to fresh smoke from a UK 2R1 reference cigarette had carboxyhemoglobin levels of 0.75 +/- 0.12 and 4.73 +/- 0.12% in sham- and smoke-exposed groups, respectively. Animals chronically exposed to cigarette smoke displayed a nearly twofold increase in pulmonary arylhydrocarbon hydroxylase activity. Smoke exposure produced a 26-33% decrease in aortic PGI2 synthesis compared to shams in the Se3E20, Se0.03E20, and Se3E0 groups. Smoking also increased platelet thromboxane 91% and 98% in the Se3E20 and Se3E0 groups compared to shams. It is concluded that cigarette-smoke exposure and selenium or vitamin E deficiency alter aortic PGI2 and platelet TXA2 production

Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

Science.gov (United States)

Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

2016-01-01

Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

Radiation-induced changes in production of prostaglandins Fsub(2. cap alpha. ), E, and thromboxane B/sub 2/ in guinea pig parenchymal lung tissues

Energy Technology Data Exchange (ETDEWEB)

Steel, L K; Catravas, G N [Armed Forces Radiobiology Research Inst., Bethesda, MD (USA)

1982-11-01

At 1 hour to 4 days after unilateral exposure of guinea pigs to a single dose (0.5, 1.5, or 3.0 Gy) of gamma-radiation, changes were detected in prostaglandin and thromboxane concentrations in parenchymal lung tissues. At 1-3 hours after exposure, tissue levels of PGFsub(2..cap alpha..), PGE, and thromboxane B/sub 2/ were significantly elevated in animals receiving 3.0 Gy, with the magnitude of alteration revealing a radiation dose effect. By 24 hours, tissue prostaglandin and thromboxane levels returned to near control values. Lung tissue synthesis of prostaglandins in response to H-1 receptor stimulation by the exogenous addition of histamine revealed similar radiation dose effects. The carboxylic acid ionophore A23187, exogenously applied to lung tissues, revealed a transient peak of increased sensitivity to ionophore stimulation for TxB/sub 2/ synthesis at 24 hours and for PGFsub(2..cap alpha..) at 72 hours post-irradiation. The data suggest that significant alterations in prostaglandin and thromboxane concentrations in parenchymal lung tissues occur following irradiation, in a dose-dependent manner, and that altered responsiveness to H-1 receptor stimulation and divalent cation transport also occur.

Effect of albumin-bound DHA on phosphoinositide phosphorylation in collagen stimulated human platelets

International Nuclear Information System (INIS)

Gaudette, D.C.; Holub, B.J.

1990-01-01

The effect of exogenous albumin-bound docosahexaenoic acid (22:6n-3) (DHA), arachidonic acid (20:4n-6) (AA), and eicosapendaenoic acid (20:5n-3) (EPA) on phosphoinositide metabolism following collagen stimulation was studied using [3H]inositol prelabelled platelets. Collagen stimulation (3 min, 1.8 micrograms/ml) increased the labelling of both phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4,5-biphosphate (PIP2). Of the fatty acids tested, only pre-incubation (2 min) with DHA (20 microM) significantly attenuated the collagen-induced increased PIP and PIP2 labelling; EPA was without effect, while AA enhanced PIP labelling. Forty microM DHA was less effective at attenuating the increased PIP and PIP2 labelling even though this concentration of DHA resulted in greater inhibition of platelet aggregation. Neither concentration of DHA attenuated the increased polyphosphoinositide labelling resulting from stimulation by the endoperoxide analogue U46619, or the phorbol ester, PMA. These data suggest that the effect of DHA on attenuating the increased PIP and PIP2 labelling following collagen stimulation likely occurs before thromboxane receptor occupancy, may not occur at the level of protein kinase C activation, and could be mediated in part via a lessened synthesis of thromboxane A2

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

Directory of Open Access Journals (Sweden)

Alexey Navdaev

Full Text Available 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.

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

Science.gov (United States)

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

Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs Formation and Arterial Thrombosis.

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Daniella M Mizurini

Full Text Available The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs, a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability.This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method.Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

Alterations in prostacyclin and thromboxane formation by chronic cigarette smoke exposure: temporal relationships and whole smoke vs. gas phase

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Lubawy, W.C.; Culpepper, B.T.; Valentovic, M.A.

1986-04-01

Chronic cigarette smoke exposure in vivo causes decreased conversion of (/sup 14/C)arachidonic acid (AA) to prostacyclin (PGI2) by isolated aortic tissue and increased conversion to thromboxane (TXA2) by isolated platelets from rats. Alterations in the PGI2/TXA2 balance may be part of the mechanism through which smoking increases the risk of cardiovascular disease. To study the influence of smoke exposure duration on this response, male rats were exposed daily to 10 puffs of freshly generated cigarette smoke. Animals were killed after 1, 4, 14, 28 and 57 days of smoke exposure and 3, 7, 14 and 28 days after cessation of the 57-day of smoke-exposure regimen. Elevated carboxyhemoglobin levels during the smoke-exposure sessions verified smoke (gas phase) inhalation. Statistically significant alterations in prostacyclin synthesis preceded those of thromboxane. A decrease of 20-25% (P less than 0.05) in PGI2 production from (/sup 14/C)AA in isolated aortic tissue was found beginning 28 days after smoke was initiated and quickly rebounded when smoke exposure was terminated. Increased production of TXA2 from (/sup 14/C)AA by isolated platelets became statistically significant (P less than 0.05) on the 57th day and returned to normal 7-14 days after cessation of smoke exposure. To determine the effect of gas phase constituents on the PGI2/TXA2 balance a second series of experiments divided male and female Sprague-Dawley rats into sham, whole smoke and gas phase groups. Gas phase was produced by passing whole smoke through a Cambridge filter to remove particulate matter. Per cent COHb averaged 1.4 for sham, 7.8 for whole smoke and 9.4 for gas phase groups.

Involvement of COX2–thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish

International Nuclear Information System (INIS)

Teraoka, Hiroki; Okuno, Yuki; Nijoukubo, Daisuke; Yamakoshi, Ayumi; Peterson, Richard E.; Stegeman, John J.; Kitazawa, Takio; Hiraga, Takeo; Kubota, Akira

2014-01-01

Highlights: • We establish a new indicator of pericardial edema in developing zebrafish (precardiac edema). • Property of precardiac edema by TCDD is similar to that for conventional pericardial edema. • COX2b (but not COX2a)–thromboxane pathway is involved in precardiac edema by TCDD. - Abstract: The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration–response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b–thromboxane

Involvement of COX2–thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish

Energy Technology Data Exchange (ETDEWEB)

Teraoka, Hiroki, E-mail: hteraoka@rakuno.ac.jp [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Okuno, Yuki; Nijoukubo, Daisuke; Yamakoshi, Ayumi [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Peterson, Richard E. [School of Pharmacy, University of Wisconsin, Madison, WI (United States); Stegeman, John J. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States); Kitazawa, Takio; Hiraga, Takeo [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Kubota, Akira [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States)

2014-09-15

Highlights: • We establish a new indicator of pericardial edema in developing zebrafish (precardiac edema). • Property of precardiac edema by TCDD is similar to that for conventional pericardial edema. • COX2b (but not COX2a)–thromboxane pathway is involved in precardiac edema by TCDD. - Abstract: The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration–response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b–thromboxane

The role of thromboxane A(2) in the pathogenesis of intrauterine growth restriction associated with maternal smoking in pregnancy.

LENUS (Irish Health Repository)

Lynch, Caoimhe M

2012-02-01

BACKGROUND: To examine the effect of maternal smoking in pregnancy on the production of two eicosanoids, thromboxane A(2) and prostacyclin I2, and their role in the pathogenesis of intrauterine growth restriction. METHODS: Prospective case control study enrolled smoking and non-smoking women at <\\/=14 weeks gestation. Maternal urine samples were obtained at <\\/=14, 28 and 36 weeks. High performance liquid chromatography tandem mass spectrometry (LC-MS-MS) was used to quantify 11-dehydrothromboxane B(2) (TX-M) and 2,3 dinor-6-ketoprostaglandin F1alpha (PG-M), stable urinary metabolites of thromboxane A(2) and prostacyclin I2. Confirmation of the smoking status was performed by quantitation of urinary nicotine metabolites. Data was analysed using SPSS and Stata((R)). RESULTS: Thirty five were enrolled in the smoking group and 32 in the non-smoking group. Smoking resulted higher levels of TX-M at <\\/=14, 28 and 36 weeks gestation. There was no difference in PG-M at any gestational time point between the two groups. The median customised birthweight centile in the smoking group was 17.0 (0-78) compared to 55.5 (4-100) in the non-smoking group (P<0.001). A causal relationship between elevated TX-M and IUGR could not be established. CONCLUSIONS: Maternal smoking in pregnancy is associated with altered eicosanoid production in favour of the vasoconstrictor thromboxane A(2) which occurs early in the first trimester.

Chronic cigarette smoke exposure adversely alters 14C-arachidonic acid metabolism in rat lungs, aortas and platelets

International Nuclear Information System (INIS)

Lubawy, W.C.; Valentovic, M.A.; Atkinson, J.E.; Gairola, G.C.

1983-01-01

Male rats were exposed to freshly generated cigarette smoke once daily, 5 times a week for 10 weeks. Inhalation of smoke was verified by elevated carboxyhemoglobin in blood sampled immediately after smoke exposure and by increased lung aryl hydrocarbon hydroxylase activity 24 hours after the last smoke exposure. Aortic rings isolated from smoke-exposed rats synthesized less prostacyclin (PGI2) from 14 C-arachidonic acid than rings from sham rats. Platelets from smoke-exposed rats synthesized more thromboxane (TXA2) from 14 C-arachidonic acid than platelets from room controls but not those from sham rats. Lung microsomes from smoke-exposed rats synthesized more TXA2 and had a lower PGI2/TXA2 ratio than lung microsomes from room controls and shams. It is concluded that chronic cigarette smoke exposure alters arachidonic acid metabolism in aortas, platelets and lungs in a manner resulting in decreased PGI2 and increased TXA2, thereby creating a condition favoring platelet aggregation and a variety of cardiovascular diseases

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

International Nuclear Information System (INIS)

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

1989-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Lin, L.; Wiesehahn, G.P.; Morel, P.A.; Corash, L. (Diamond Scientific Company, Des Moines, IA (USA))

1989-07-01

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

In vitro model of platelet aggregation in stenotic arteries

International Nuclear Information System (INIS)

Morley, D.; Santamore, W.P.

1988-01-01

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

The impact of night-shift work on platelet function in healthy medical staff.

Science.gov (United States)

Nakao, Tomoko; Yasumoto, Atsushi; Tokuoka, Suzumi; Kita, Yoshihiro; Kawahara, Takuya; Daimon, Masao; Yatomi, Yutaka

2018-04-18

Rotating shift work has been reported to increase the risk of cardiovascular diseases. Vascular endothelial dysfunction and platelet activation are among the leading causes of thrombus formation in patients with myocardial infarction or stroke. Endothelial function has been shown to be impaired immediately after night-shift work; however, it is not known whether platelets are also activated. The aim of this study was to investigate the acute impact of night-shift work on platelet function. This observational study included 11 healthy medical staff members (seven women, median age 32 years). We examined each subject's platelet aggregation rates and the serum concentrations of eicosanoid mediators after night-shift work and on day-shift work without preceding night-shift work (baseline). Platelet aggregation did not differ from baseline levels after night-shift work. However, serum cyclooxygenase (COX)-metabolized eicosanoid mediators, particularly thromboxane (Tx) B 2 (a stable metabolite of TxA 2 and the most important marker of platelet activation), were significantly higher after the night-shift than at baseline (median 65.3 vs 180.4 ng/ml). Although platelet aggregation did not increase, there was an increase in serum COX-metabolized eicosanoid mediators such as TxB 2 in healthy medical staff after night-shift work. This platelet hypersensitivity may be one of the mechanisms underlying the significant association between night-shift work and adverse cardiovascular outcomes.

The C50T polymorphism of the cyclooxygenase-I gene and the risk of thrombotic events during low-dose therapy with acetyl salicylic acid

NARCIS (Netherlands)

Clappers, Nick; van Oijen, Martijn G. H.; Sundaresan, Santosh; Brouwer, Marc A.; te Morsche, Rene H. M.; Keuper, Wessel; Peters, Wilbert H. M.; Drenth, Joost P. H.; Verheugt, Freek W. A.

2008-01-01

prevents thrombotic events by inhibiting platelet cyclooxygenase-I (COX-1), thus reducing thromboxane A2 formation and platelet aggregation.The C50T polymorphism of COX-1 is associated with an impaired inhibition of both thromboxane production and in-vitro platelet aggregation by aspirin.We studied

Thromboxane synthesis inhibitors and postprandial jejunal capillary exchange capacity.

Science.gov (United States)

Mangino, M J; Chou, C C

1988-05-01

The effects of thromboxane synthesis inhibitors (imidazole and U 63557A; Upjohn) and the cyclooxygenase inhibitor, mefenamic acid, on jejunal capillary filtration coefficients (Kfc) were determined in dogs before and during the presence of predigested food in the jejunal lumen. The jejunal Kfc increased significantly soon after the placement of a predigested test food containing all major constituents of diet. The Kfc remained elevated as long as the food was present in the lumen (15 min). Mefenamic acid (10 mg/kg iv) did not significantly alter resting jejunal Kfc or alter the food-induced increase in Kfc. Imidazole (5.0 mg/min ia) or U 63557A (5.0 mg/kg iv) per se significantly increased jejunal Kfc. Placement of digested food further increased the Kfc to levels significantly higher than those observed before administration of the two thromboxane synthase inhibitors. Production of thromboxane B2 by jejunal tissue was significantly reduced and 6-ketoprostaglandin F1 alpha (the stable hydrolysis product of prostacyclin) production was significantly increased after administration of U 63557A. Our study indicates that the relative production of endogenous thromboxanes and other prostanoids modulates jejunal capillary exchange capacity in the absence or presence of digested food in the jejunal lumen.

Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

Directory of Open Access Journals (Sweden)

Hou Ssu-Yu

2010-06-01

Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

Modulation of platelet aggregation by areca nut and betel leaf ingredients: roles of reactive oxygen species and cyclooxygenase.

Science.gov (United States)

Jeng, Jiiang-Huei; Chen, Shiao-Yun; Liao, Chang-Hui; Tung, Yuan-Yii; Lin, Bor-Ru; Hahn, Liang-Jiunn; Chang, Mei-Chi

2002-05-01

There are 2 to 6 billion betel quid (BQ) chewers in the world. Areca nut (AN), a BQ component, modulates arachidonic acid (AA) metabolism, which is crucial for platelet function. AN extract (1 and 2 mg/ml) stimulated rabbit platelet aggregation, with induction of thromboxane B2 (TXB2) production. Contrastingly, Piper betle leaf (PBL) extract inhibited AA-, collagen-, and U46619-induced platelet aggregation, and TXB2 and prostaglandin-D2 (PGD2) production. PBL extract also inhibited platelet TXB2 and PGD2 production triggered by thrombin, platelet activating factor (PAF), and adenosine diphosphate (ADP), whereas little effect on platelet aggregation was noted. Moreover, PBL is a scavenger of O2(*-) and *OH, and inhibits xanthine oxidase activity and the (*)OH-induced PUC18 DNA breaks. Deferoxamine, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and neomycin prevented AN-induced platelet aggregation and TXB2 production. Indomethacin, genistein, and PBL extract inhibited only TXB2 production, but not platelet aggregation. Catalase, superoxide dismutase, and dimethylthiourea (DMT) showed little effect on AN-induced platelet aggregation, whereas catalase and DMT inhibited the AN-induced TXB2 production. These results suggest that AN-induced platelet aggregation is associated with iron-mediated reactive oxygen species production, calcium mobilization, phospholipase C activation, and TXB2 production. PBL inhibited platelet aggregation via both its antioxidative effects and effects on TXB2 and PGD2 production. Effects of AN and PBL on platelet aggregation and AA metabolism is crucial for platelet activation in the oral mucosa and cardiovascular system in BQ chewers.

Characterization of a thromboxane A2/prostaglandin H2 receptor in guinea pig lung membranes using a radioiodinated thromboxane mimetic

International Nuclear Information System (INIS)

Saussy, D.L. Jr.; Mais, D.E.; Dube, G.P.; Magee, D.E.; Brune, K.A.; Kurtz, W.L.; Williams, C.M.

1991-01-01

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent constrictors of airway smooth muscle and may mediate some of the pulmonary effects of leukotrienes. To date, the TXA2/PGH2 receptor in lung has not been well characterized. In this report, we describe the evaluation of the TXA2/PGH2 receptor in guinea pig lung membranes using the new radiolabeled TXA2 mimetic [1S(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha)]-7-[3-(3-hydroxy-4-(4'- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-h eptenoic acid (IBOP). IBOP elicited a dose-dependent contraction of guinea pig lung parenchymal strips (EC50 = 3.03 +/- 0.97 nM, three experiments), which was blocked by the TXA2/PGH2 antagonists SQ29548 (pKB = 7.44 +/- 0.2, three experiments), BM13505 (pKB = 6.29 +/- 0.26, three experiments), and I-PTA-OH (pKB = 5.82 +/- 0.36, three experiments). In radioligand binding studies, the binding of [125I]IBOP to guinea pig lung membranes prepared from perfused lungs was saturable, displaceable, and dependent upon protein concentration. Binding was optimal at pH 6.5 and was enhanced by the addition of mono- and divalent cations. The standard assay buffer was 25 mM 3-(N-morpholino)propanesulfonic acid, pH 6.5, 100 mM NaCl, 5 mM MgCl2. Binding was inhibited by pretreatment with dithiothreitol, N-ethylmaleimide, or beta-mercaptoethanol. Binding was unaffected by the addition of guanine nucleotide analogs at concentrations up to 300 microM. Analysis of the time course of binding of [125]IBOP at 30 degrees yielded k-1 = 0.0447 min-1, k1 = 2.49 x 10(8) M-1 min-1, and Kd = k-1/k1 = 180 pM. Computer analysis of equilibrium binding studies using nonlinear methods (LUNDON-1) revealed a single class of noninteracting binding sites with a Kd of 86.9 +/- 11.9 pM and a Bmax of 81.8 +/- 7.7 fmol/mg of protein (three experiments)

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

Science.gov (United States)

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.

Antiplatelet Effects of Qishen Yiqi Dropping Pill in Platelets Aggregation in Hyperlipidemic Rabbits

Directory of Open Access Journals (Sweden)

Yi Wang

2012-01-01

Full Text Available We investigated the effects of Qishen Yiqi Dropping Pill (QSYQ on platelets aggregation and its possible mechanisms. Hyperlipidemic model in rabbits was produced by a high fat/cholesterol diet for 6 weeks, the therapeutic effect of QSYQ with 2.0 g/kg, 1.0 g/kg, and 0.5 g/kg was observed. Fourteen days after drug treatment, platelet aggregation induced by adenosine diphosphate (ADP, arachidonic acid (AA, and collagen (COLL was significantly reduced in rabbits of model group. Moreover, β-thromboglobulin (β-TG level decreased obviously but no significant change in P-selectin and platelet factor 4 (PF4 level, while QSYQ significantly decreased the ratio of thromboxane B2 (TXB2 to 6-keto-prostaglandin F1α (6-Keto-PGF1α and increased cyclic adenosine monophosphate (cAMP level in rabbits. In summary, QSYQ can improve platelets aggregation and inhibit the over-release of β-TG in hyperlipidemic rabbits; and the increased cAMP level may be involved in this process. These results suggest that the antiplatelet aggregation effect of QSYQ may be due to its ability to increase cAMP level for improving cAMP metabolism.

Study of exon 12 polymorphism of the human thromboxane synthase (CYP5A1) gene in Egyptian stroke patients

International Nuclear Information System (INIS)

Soliman, S.E.T.; Zaater, M.K.

2010-01-01

Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed screening for the prevalence of exon12 polymorphism of the human Thromboxane synthase (CYP5A1) gene among Egyptian normal and stroke patients. Using sequence-specific PCR, we examined the allelic prevalence in 70 Egyptian patients with ischemic strokes and in 70 controls. In addition, we compared the CYP5A1 allelic prevalence in 30 patients with stroke recurrence despite Aspirin use, in comparison with patients who have not experienced recurrent stroke while taking Aspirin. The frequencies of the CYP5A1*9 mutant (substitution of guanine by adenine near the heme-binding catalytic domain) and of the wild-type allele were 0.197(19.7%) and 0.803 (80.3%) respectively; they did not differ significantly between stroke patients and controls. The CYP5A1*9 mutant was significantly more prevalent among stroke patients with history of previous cerebrovascular attacks; even after adjusting for the common risk factors for cardiovascular disease (odds ratio (OR)1.73, 95%, confidence interval ( CI) 1.10-2.73; p=0.017). Among stroke patients, the presence of the CYP5A1 wild type allele was more frequent among the hypertensives (OR 1.68, 95% CI, 1.01-2.79; p=0.045), and less frequent among the diabetics (OR 0.55, 95%, CI 0.36-0.84; p=0.006). Also among stroke patients, the CYP5A1*9 mutant was significantly more prevalent among those, who failed secondary Aspirin prophylaxis compared to those with successful secondary Aspirin prophylaxis (OR 1.49, 95%, CI 1.06-2.11). This study provides evidence for high prevalence of the CYP5A1*9 mutant

Semiquantitative dynamic computed tomography to predict response to anti-platelet therapy in acute cerebral infarction

International Nuclear Information System (INIS)

Chokyu, K.; Shimizu, K.; Fukumoto, M.; Mori, T.; Mokudai, T.; Mori, K.

2002-01-01

We investigated whether dynamic computed tomography (CT) in patients with acute cerebral infarction could identify patients likely to respond to anti-platelet therapy. Seventy patients underwent semiquantitative dynamic CT within 6 h as well as cerebral angiography. All then received anti-platelet therapy with a thromboxane A2 synthetase inhibitor. Peak value (pv) and time-to-peak (tp) (time-density curves) for the Sylvian fissure were extracted from dynamic CT data and standardizing interpatient data, two indices, PV/TP index and TP index, were prepared following a standard semiquantitative manner. Both PV/TP index and TP index were effective in discriminating between 48 responders (modified Rankin scale (mRS): 0 to 2) and 22 non-responders (mRS: 3 to 5, or death: 6; both P 1.1) and non-compensated rCBF. Intermediate PV/TP values could not predict outcome. Dynamic CT prior to therapy can identify patients with acute cerebral infarction who are treatable with anti-platelet therapy alone. (orig.)

Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans.

Science.gov (United States)

Song, Wen-Liang; Stubbe, Jane; Ricciotti, Emanuela; Alamuddin, Naji; Ibrahim, Salam; Crichton, Irene; Prempeh, Maxwell; Lawson, John A; Wilensky, Robert L; Rasmussen, Lars Melholt; Puré, Ellen; FitzGerald, Garret A

2012-04-01

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Plasma thromboxane B2 levels in horses experimentally infected with Strongylus vulgaris.

Science.gov (United States)

Cambridge, H; Reynoldson, J A; Dunsmore, J D

1989-06-01

Plasma thromboxane B2 (TXB2) the stable inactive metabolite of thromboxane A2 (TXA2), was measured daily by specific radioimmunoassay in three groups of animals before and after experimental infection with Strongylus vulgaris. Infection of four 'parasite naive' foals produced a typical acute syndrome with intermittent but statistically insignificant rises in TXB2 levels. Interpretation of results was complicated by the presence of a non-septic peritonitis associated with implantation of the foals with electrodes for recording myoelectrical activity. In two foals of similar age, with some natural exposure to S. vulgaris, there was little or no clinical response to infection and increases in TXB2 were absent. Baseline levels were also much lower, indicating that the peritonitis may have affected the results obtained in the first group of foals. Severe mesenteric arteritis was confirmed at necropsy in all six foals. A third group of yearling horses, all with natural exposure to the parasite, were generally resistant to infection. One animal developed arteritis with clinical signs of diarrhoea and mild colic, and also showed intermittent increases in TXB2. The mean plasma TXB2 level after infection was significantly higher than in the control period, although absolute levels were lower than those recorded in the 'parasite naive' foals. Other animals in this group had low TXB2 levels and minimal arteritis was found at necropsy. These results indicate that although infection appears to have an effect on plasma TXB2, the changes are inconsistent and not reliable indicators of the presence of verminous arteritis. The results also confirm the difficulty in establishing infection and the variability of the response in animals with previous exposure.

Examination of thromboxane synthase as a prognostic factor and therapeutic target in non-small cell lung cancer.

LENUS (Irish Health Repository)

Cathcart, Mary-Clare

2011-03-01

Thromboxane synthase (TXS) metabolises prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with a poor prognosis. TXS inhibition induces cell death in-vitro, providing a rationale for therapeutic intervention. We aimed to determine the expression profile of TXS in NSCLC and if it is prognostic and\\/or a survival factor in the disease.

Effects of single oral doses of lysine clonixinate and acetylsalicylic acid on platelet functions in man.

Science.gov (United States)

Pallapies, D; Muhs, A; Bertram, L; Rohleder, G; Nagyiványi, P; Peskar, B A

1996-01-01

Lysine clonixinate is an analgesic drug with a so far unknown mechanism of action. We have determined its effect on platelet cyclooxygenase in man. Biosynthesis of thromboxane (TX)B2 and prostaglandin (PG)F2 alpha in clotting whole blood ex vivo as well as collagen-induced platelet aggregation measured before and at various time points after oral administration of 125 mg lysine clonixinate were compared to results obtained with 500 mg acetylsalicylic acid (ASA). While biosynthesis of both TXB2 and PGF2 alpha measured radioimmunologically was inhibited significantly 2.5 h, but not 6 h, after administration of lysine clonixinate, inhibition by ASA was much greater and still highly significant after 48 h. Similarly, collagen-induced aggregation of platelet-rich plasma was inhibited for a longer period and to a greater extent after administration of ASA than after lysine clonixinate. Our results indicate that lysine clonixinate is a cyclooxygenase inhibitor of moderate potency. It remains to be investigated whether mechanisms other than inhibition of cyclooxygenase contribute to the analgesic activity of lysine clonixinate.

Effect of Antrodia camphorata on Inflammatory Arterial Thrombosis-Mediated Platelet Activation: The Pivotal Role of Protein Kinase C

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Wan-Jung Lu

2014-01-01

Full Text Available Antrodia camphorata is a rare Taiwanese medicinal mushroom. Antrodia camphorata extract has been reported to exhibit antioxidant, anti-inflammation, antimetastasis, and anticancer activities and plays a role in liver fibrosis, vasorelaxation, and immunomodulation. Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Platelet activation plays a crucial role in intravascular thrombosis, which is involved in a wide variety of cardiovascular diseases. However, the effect of Antrodia camphorata on platelet activation remains unclear. We examined the effects of Antrodia camphorata on platelet activation. In the present study, Antrodia camphorata treatment (56–224 μg/mL inhibited platelet aggregation induced by collagen, but not U46619, an analogue of thromboxane A2, thrombin, and arachidonic acid. Antrodia camphorata inhibited collagen-induced calcium (Ca2+ mobilization and phosphorylation of protein kinase C (PKC and Akt. In addition, Antrodia camphorata significantly reduced the aggregation and phosphorylation of PKC in phorbol-12, 13-dibutyrate (PDBu activated platelets. In conclusion, Antrodia camphorata may inhibit platelet activation by inhibiting of Ca2+ and PKC cascade and the Akt pathway. Our study suggests that Antrodia camphorata may be a potential therapeutic agent for preventing or treating thromboembolic disorders.

Inhibitory Effect of the Punica granatum Fruit Extract on Angiotensin-II Type I Receptor and Thromboxane B2 in Endothelial Cells Induced by Plasma from Preeclamptic Patients.

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Kusumawati, Widya; Keman, Kusnarman; Soeharto, Setyawati

2016-01-01

This study aims to evaluate whether the Punica granatum fruit extract modulates the Angiotensin-II Type I receptor (AT1-R) and thromboxane B2 level in endothelial cells induced by plasma from preeclamptic patients. Endothelial cells were obtained from human umbilical vascular endothelial cells. At confluence, endothelial cells were divided into five groups, which included endothelial cells exposed to 2% plasma from normal pregnancy (NP), endothelial cells exposed to 2% plasma from preeclamptic patients (PP), and endothelial cells exposed to PP in the presence of ethanolic extract of Punica granatum (PP + PG) at the following three doses: 14; 28; and 56 ppm. The expression of AT1-R was observed by immunohistochemistry technique, and thromboxane B2 level was done by immunoassay technique. Plasma from PP significantly increased AT1-R expression and thromboxane B2 levels compared to cells treated by normal pregnancy plasma. The increasing of AT1-R expression significantly (P Punica granatum extract. Moreover, the increasing of thromboxane B2 levels significantly (P Punica granatum extract. We further concluded that Punica granatum fruit protects and inhibits the sensitivity of endothelial cells to plasma from preeclamptic patients due to inhibition of AT1-R expression (56 ppm) and reduced thromboxane B2 levels (14 ppm).

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

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Lordkipanidzé, Marie; Lowe, Gillian C; Kirkby, Nicholas S; Chan, Melissa V; Lundberg, Martina H; Morgan, Neil V; Bem, Danai; Nisar, Shaista P; Leo, Vincenzo C; Jones, Matthew L; Mundell, Stuart J; Daly, Martina E; Mumford, Andrew D; Warner, Timothy D; Watson, Steve P

2014-02-20

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

Up-regulation of thromboxane A2 receptor expression by lipid soluble smoking particles through post-transcriptional mechanisms

DEFF Research Database (Denmark)

Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

2008-01-01

Atherosclerosis is a key factor in vascular disease, and cigarette smoking is a well-known risk factor that may induce an inflammatory response and enhance plaque formation in arteries. Thromboxane (Tx) is one key inflammatory mediator involved in the pathogenesis of cardiovascular disease....... The present study was designed to test if lipid soluble smoking particles (DSP) enhance TxA(2) receptor (TP) expression in rat mesenteric arteries, and if intracellular mitogen-activated protein kinase (MAPK) pathways play a role. Organ culture of rat mesenteric arteries in the presence of DSP (0.2 microl...

Effects of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs.

Science.gov (United States)

Blois, Shauna L; Allen, Dana G; Wood, R Darren; Conlon, Peter D

2010-03-01

To determine effects of therapeutic dosages of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs. 10 hound-crossbred dogs. Aspirin (10 mg/kg, PO, q 12 h), carprofen (4.4 mg/kg, PO, q 24 h), deracoxib (2 mg/kg, PO, q 24 h), meloxicam (0.1 mg/kg, PO, q 24 h), and a placebo were administered for 7 days in a random order to each of 10 healthy dogs; there was a 21-day washout period between subsequent treatments. One-stage prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and plasma concentrations of thromboxane (TX)B(2) and 6-keto prostaglandin (PG)F(1alpha) were measured before and after treatment administration. Platelet function was assessed by use of a platelet-function analyzer and aggregation. Aspirin, carprofen, and meloxicam did not significantly affect platelet function. Deracoxib caused a mild decrease in platelet aggregation induced by 50microM ADP. Platelet number, Hct, PT, aPTT, and plasma TXB(2) and 6-keto PGF(1alpha) concentrations were unchanged after NSAID administration. Meloxicam administration resulted in a significant decrease in fibrinogen concentration, but results remained within the laboratory reference interval. Oral administration of commonly used NSAIDs at therapeutic dosages in healthy dogs did not alter plasma TXB(2) and 6-keto PGF(1alpha) concentrations. Deracoxib administration resulted in a minor abnormality in platelet aggregation. Anti-inflammatory doses of aspirin did not affect platelet function as measured by use of optical aggregometry and a platelet-function analyzer. Further evaluation of the effects of aspirin and cyclooxygenase-2-selective inhibitors on hemostasis should be performed.

Acetylsalicylic Acid Daily vs Acetylsalicylic Acid Every 3 Days in Healthy Volunteers: Effect on Platelet Aggregation, Gastric Mucosa, and Prostaglandin E2 Synthesis.

Science.gov (United States)

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. © 2015, The American College of Clinical Pharmacology.

Superoxide Dismutase 2 is dispensable for platelet function.

Science.gov (United States)

Fidler, Trevor P; Rowley, Jesse W; Araujo, Claudia; Boudreau, Luc H; Marti, Alex; Souvenir, Rhonda; Dale, Kali; Boilard, Eric; Weyrich, Andrew S; Abel, E Dale

2017-10-05

Increased intracellular reactive oxygen species (ROS) promote platelet activation. The sources of platelet-derived ROS are diverse and whether or not mitochondrial derived ROS, modulates platelet function is incompletely understood. Studies of platelets from patients with sickle cell disease, and diabetes suggest a correlation between mitochondrial ROS and platelet dysfunction. Therefore, we generated mice with a platelet specific knockout of superoxide dismutase 2 (SOD2-KO) to determine if increased mitochondrial ROS increases platelet activation. SOD2-KO platelets demonstrated decreased SOD2 activity and increased mitochondrial ROS, however total platelet ROS was unchanged. Mitochondrial function and content were maintained in non-stimulated platelets. However SOD2-KO platelets demonstrated decreased mitochondrial function following thrombin stimulation. In vitro platelet activation and spreading was normal and in vivo, deletion of SOD2 did not change tail-bleeding or arterial thrombosis indices. In pathophysiological models mediated by platelet-dependent immune mechanisms such as sepsis and autoimmune inflammatory arthritis, SOD2-KO mice were phenotypically identical to wildtype controls. These data demonstrate that increased mitochondrial ROS does not result in platelet dysfunction.

Radioimmunological thromboxane concentrations in arterial blood of cigarette smokers and hypertensive subjects

International Nuclear Information System (INIS)

Krause, F.J.

1987-01-01

Thromboxane concentrations were investigated in the arterial plasma of 79 patients (24 smokers, 23 nonsmokers, 16 hypertensives, 16 normotensives) with periphered obstructive arterial disease. The thromboxane investigation was made by radioimmunoassay, whose realization was modified. The results indicate that nonsmokers have significantly lower thromboxane levels than smokers, whereas there is no statistically significant difference between hypertensives and normotensives. The findings coincide with the increased risk for arteriosclerosis of smokers, that doesn't work for hypertensives. (orig.) [de

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

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Philip M Sobolesky

Full Text Available The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-β receptor (TPβ, but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPβ raised basal phosphorylated (pFOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619. Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2 or ERK inhibitor (U0126. Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27

Energy Technology Data Exchange (ETDEWEB)

Leung, Kin Chung; Hsin, Michael K.Y.; Chan, Joey S.Y.; Yip, Johnson H.Y.; Li, Mingyue; Leung, Billy C.S. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Mok, Tony S.K. [Department of Clinical Oncology, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Warner, Timothy D. [The William Harvey Research Institute, Queen Mary University of London, London (United Kingdom); Underwood, Malcolm J. [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong); Chen, George G., E-mail: gchen@cuhk.edu.hk [Department of Surgery, The Chinese University of Hong Kong, Shatin, New Territories (Hong Kong)

2009-10-15

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB{sub 2}) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB{sub 2} but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.

Inhibition of thromboxane synthase induces lung cancer cell death via increasing the nuclear p27

International Nuclear Information System (INIS)

Leung, Kin Chung; Hsin, Michael K.Y.; Chan, Joey S.Y.; Yip, Johnson H.Y.; Li, Mingyue; Leung, Billy C.S.; Mok, Tony S.K.; Warner, Timothy D.; Underwood, Malcolm J.; Chen, George G.

2009-01-01

The role of thromboxane in lung carcinogenesis is not clearly known, though thromboxane B2 (TXB 2 ) level is increased and antagonists of thromboxane receptors or TXA2 can induce apoptosis of lung cancer cells. p27, an atypical tumor suppressor, is normally sequestered in the nucleus. The increased nuclear p27 may result in apoptosis of tumor cells. We hypothesize that the inhibition of thromboxane synthase (TXS) induces the death of lung cancer cells and that such inhibition is associated with the nuclear p27 level. Our experiment showed that the inhibition of TXS significantly induced the death or apoptosis in lung cancer cells. The activity of TXS was increased in lung cancer. The nuclear p27 was remarkably reduced in lung cancer tissues. The inhibition of TXS caused the cell death and apoptosis of lung cancer cells, likely via the elevation of the nuclear p27 since the TXS inhibition promoted the nuclear p27 level and the inhibition of p27 by its siRNA recovered the cell death induced by TXS inhibition. Collectively, lung cancer cells produce high levels of TXB 2 but their nuclear p27 is markedly reduced. The inhibition of TXS results in the p27-related induction of cell death in lung cancer cells.

Ozagrel hydrochloride, a selective thromboxane A2 synthase inhibitor, alleviates liver injury induced by acetaminophen overdose in mice

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Tomishima Yoshiro

2013-01-01

Full Text Available Abstract Background Overdosed acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP causes severe liver injury. We examined the effects of ozagrel, a selective thromboxane A2 (TXA2 synthase inhibitor, on liver injury induced by APAP overdose in mice. Methods Hepatotoxicity was induced to ICR male mice by an intraperitoneal injection with APAP (330 mg/kg. The effects of ozagrel (200 mg/kg treatment 30 min after the APAP injection were evaluated with mortality, serum alanine aminotransferase (ALT levels and hepatic changes, including histopathology, DNA fragmentation, mRNA expression and total glutathione contents. The impact of ozagrel (0.001-1 mg/mL on cytochrome P450 2E1 (CYP2E1 activity in mouse hepatic microsome was examined. RLC-16 cells, a rat hepatocytes cell line, were exposed to 0.25 mM N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite of APAP. In this model, the cytoprotective effects of ozagrel (1–100 muM were evaluated by the WST-1 cell viability assay. Results Ozagel treatment significantly attenuated higher mortality, elevated serum alanine aminotransferase levels, excessive hepatic centrilobular necrosis, hemorrhaging and DNA fragmentation, as well as increase in plasma 2,3-dinor thromboxane B2 levels induced by APAP injection. Ozagrel also inhibited the hepatic expression of cell death-related mRNAs induced by APAP, such as jun oncogene, FBJ osteosarcoma oncogene (fos and C/EBP homologous protein (chop, but did not suppress B-cell lymphoma 2-like protein11 (bim expression and hepatic total glutathione depletion. These results show ozagrel can inhibit not all hepatic changes but can reduce the hepatic necrosis. Ozagrel had little impact on CYP2E1 activity involving the NAPQI production. In addition, ozagrel significantly attenuated cell injury induced by NAPQI in RLC-16. Conclusions We demonstrate that the TXA2 synthase inhibitor, ozagrel, dramatically alleviates liver injury induced by APAP in mice, and suggest

High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

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Bing Xu

Full Text Available G protein-coupled receptors (GPCRs exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(--TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

Influence of Garlic Oil and/or Taurine Intake on Modulating the Quantity and Quality of Murine Platelets

International Nuclear Information System (INIS)

Amer, M.M.

2010-01-01

Several studies have demonstrated that enhanced platelets reactivity associated with hyperlipidaemia plays a role in the formation of atherosclerosis. Thus, platelet count, platelet aggregation in response to the agonists ADP, collagen and thromboxan B2 (TxB 2 ) were investigated in the normal and hyperlipidaemic rats to elucidate the role of some antioxidants, as modulating factors in the development of atherosclerosis. Serum concentrations of the major lipid classes, thyroid hormones, serum glutathione as well as platelet count and aggregation were determined in normal lipidemic rats (control) and compared with their corresponding levels in hyperlipidaemic ones. Hyperlipidaemia was induced by feeding rats with diet enriched with 20 g cow milk butter/100 g diet for 8 weeks. Garlic oil (200 mg/kg b.w.) and/or taurine (500 ml/kg b.w.) were peritoneal injected from the beginning of the 9th week for 15 consecutive days. Incorporation of extra fat in diet of rats for long term led to a significant (p 2 levels. On the other hand, HDL-cholesterol, serum glutathione (GSH), and T 3 and T 4 hormones level were significantly (p 2 level also showed a significant decrease. Conversely, serum HDL, T 3 , T 4 and GSH levels were significantly increased. These results denote that garlic oil and/or taurine may decrease the risk of atherosclerosis and thrombus formation associated with hyperlipidaemia through modulating the quantity and quality of platelets in male rats

Blood rheology of angina pectoris patients with myocardial injury after ischemia reperfusion and its effect on thromboxane B2 levels.

Science.gov (United States)

Wang, Wenlong; Huang, Xiaohui; Sun, Yiyong; Zhang, Jinying

2018-01-01

This study investigated the changes in the blood rheology of patients with angina pectoris and ischemia reperfusion injury and their effect on thromboxane B 2 (TXB 2 ) levels to examine their relationship. Forty patients with unstable angina pectoris who underwent elective percutaneous coronary intervention (PCI) were selected for the unstable angina group (UA group) and forty patients deemed free of coronary heart disease by coronary angiography were selected for the control group. Venous blood samples were drawn from all participants; patients in the UA group had blood drawn 1 day before and 1 day after the PCI procedure. Blood samples were used to analyze blood rheology and examine hemodynamic parameters, at the same time radioimmunoassay was applied to measure the concentrations of serum endothelin-1 (ET-1) and TXB 2 , and an automatic biochemical analyzer was used to detect the content of superoxide dismutase (SOD) and malondialdehyde (MDA). Our results showed the patients in the UA group all presented hyperviscosity; however the levels were higher for the patients in the UA group (after surgery) than for those in the UA group (before surgery). Patients in the control group exhibited normal levels, and the differences among groups were significant in pairwise comparisons (Pangina pectoris and ischemia reperfusion injury. The higher than normal TXB 2 levels can be used as a marker of platelet activation and a reference for clinical risk stratification, thus having great significance for the prevention and treatment of ischemia reperfusion injury and assessment of disease progression.

Thromboxane plays a role in postprandial jejunal oxygen uptake and capillary exchange.

Science.gov (United States)

Alemayehu, A; Chou, C C

1990-09-01

The effects of a thromboxane A2 (TxA2)-endoperoxide receptor antagonist, SQ 29548, on jejunal blood flow, oxygen uptake, and capillary filtration coefficient (Kfc) were determined in anesthetized dogs under resting conditions and during the presence of predigested food in the jejunal lumen in three series of experiments. In series 1, 2.0 micrograms intra-arterial administration of SQ 29548 was found to abolish completely the vasoconstrictor action of graded doses (0.05-2.0 micrograms) of intra-arterial injection of a TxA2-endoperoxide analogue, U44069. SQ 29548 (2.0 micrograms ia) per se did not significantly alter resting jejunal blood flow, oxygen uptake, capillary pressure, or Kfc. Before SQ 29548, placement of food plus bile into the jejunal lumen increased blood flow +42 +/- 9%, oxygen uptake +28 +/- 7%, and Kfc +24 +/- 6%. After SQ 29548, the food placement increased blood flow +37 +/- 8%, oxygen uptake +52 +/- 11%, and Kfc +63 +/- 20%. The food-induced increases in oxygen uptake and Kfc after SQ 29548 were significantly greater than those induced before the blocking of TxA2-endoperoxide receptors by SQ 29548. Our study indicates that endogenous thromboxane does not play a role in regulating jejunal blood flow, capillary filtration, and oxygen uptake under resting conditions. However, it plays a role in limiting the food-induced increases in jejunal oxygen uptake and capillary exchange capacity without influencing the food-induced hyperemia.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

International Nuclear Information System (INIS)

Petri, Marcelo H.; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

2013-01-01

Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A 2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Petri, Marcelo H. [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Tellier, Céline; Michiels, Carine [NARILIS, URBC, University of Namur, Namur (Belgium); Ellertsen, Ingvill [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden); Dogné, Jean-Michel [Department of Pharmacy, Namur Thrombosis and Hemostasis Center, University of Namur, Namur (Belgium); Bäck, Magnus, E-mail: Magnus.Back@ki.se [Department of Medicine, Karolinska Institutet and Center for Molecular Medicine, Karolinska University Hospital, Stockholm (Sweden)

2013-11-15

Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo

International Nuclear Information System (INIS)

Teraoka, Hiroki; Kubota, Akira; Dong, Wu; Kawai, Yusuke; Yamazaki, Koji; Mori, Chisato; Harada, Yoshiteru; Peterson, Richard E.; Hiraga, Takeo

2009-01-01

Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic vein blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo

Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

OpenAIRE

Jarvis, Gavin E.; Best, Denise; Watson, Steve P.

2004-01-01

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induc...

Examination of thromboxane synthase as a prognostic factor and therapeutic target in non-small cell lung cancer

LENUS (Irish Health Repository)

Cathcart, Mary-Clare

2011-03-09

Abstract Background Thromboxane synthase (TXS) metabolises prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with a poor prognosis. TXS inhibition induces cell death in-vitro, providing a rationale for therapeutic intervention. We aimed to determine the expression profile of TXS in NSCLC and if it is prognostic and\\/or a survival factor in the disease. Methods TXS expression was examined in human NSCLC and matched controls by western analysis and IHC. TXS metabolite (TXB2) levels were measured by EIA. A 204-patient NSCLC TMA was stained for COX-2 and downstream TXS expression. TXS tissue expression was correlated with clinical parameters, including overall survival. Cell proliferation\\/survival and invasion was examined in NSCLC cells following both selective TXS inhibition and stable TXS over-expression. Results TXS was over-expressed in human NSCLC samples, relative to matched normal controls. TXS and TXB2 levels were increased in protein (p < 0.05) and plasma (p < 0.01) NSCLC samples respectively. TXS tissue expression was higher in adenocarcinoma (p < 0.001) and female patients (p < 0.05). No significant correlation with patient survival was observed. Selective TXS inhibition significantly reduced tumour cell growth and increased apoptosis, while TXS over-expression stimulated cell proliferation and invasiveness, and was protective against apoptosis. Conclusion TXS is over-expressed in NSCLC, particularly in the adenocarcinoma subtype. Inhibition of this enzyme inhibits proliferation and induces apoptosis. Targeting thromboxane synthase alone, or in combination with conventional chemotherapy is a potential therapeutic strategy for NSCLC.

The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14C]arachidonic acid and [14C]eicosapentaenoic acid by human platelets

International Nuclear Information System (INIS)

Hirai, A.; Terano, T.; Hamazaki, T.

1982-01-01

It has been suggested by several investigators that eicosapentaenoic acid (C20:5 omega 3, EPA) might have anti-thrombotic effects. In this experiment, the effect of the oral administration of EPA rich fish oil concentrate on platelet aggregation and the release and the metabolism of [ 1 - 14 C]arachidonic acid and [(U)- 14 C]eicosapentaenoic acid by human platelets was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4 g) per day for 4 weeks. Plasma and platelet concentrations of EPA markedly increased, while those of arachidonic acid (C20:4 omega 6, AA) and docosahexaenoic acid (C22:6 omega 3, DHA) did not change. Platelet aggregation induced by collagen and ADP was reduced. Collagen induced [ 14 C]thromboxane B2 (TXB2) formation from [ 14 C]AA prelabeled platelets decreased. There was no detectable formation of [ 14 C]TXB3 from [ 14 C]EPA prelabeled platelets, and the conversion of exogenous [ 14 C]EPA to [ 14 C]TXB3 was lower than that of [ 14 C]AA to [ 14 C]TXB2. The release of [ 14 C]AA from [ 14 C]AA prelabeled platelets by collagen was significantly decreased. These observations raise the possibility that the release of arachidonic acid from platelet lipids might be affected by the alteration of EPA content in platelets

Intracellular Erythrocyte Platelet-activating Factor Acetylhydrolase I Inactivates Aspirin in Blood*

Science.gov (United States)

Zhou, Gang; Marathe, Gopal K.; Willard, Belinda; McIntyre, Thomas M.

2011-01-01

Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A2 with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A2 synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood. PMID:21844189

Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood.

Science.gov (United States)

Zhou, Gang; Marathe, Gopal K; Willard, Belinda; McIntyre, Thomas M

2011-10-07

Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.

Morusinol extracted from Morus alba inhibits arterial thrombosis and modulates platelet activation for the treatment of cardiovascular disease.

Science.gov (United States)

Lee, Jung-Jin; Yang, Hyun; Yoo, Yeong-Min; Hong, Seong Su; Lee, Dongho; Lee, Hyun-Jung; Lee, Hak-Ju; Myung, Chang-Seon; Choi, Kyung-Chul; Jeung, Eui-Bae

2012-01-01

Morus alba (white mulberry) has been used in traditional Chinese medicine as an anti-headache, diuretic, expectorant, and anti-diabetic agent. In previous studies, extracts of Morus alba demonstrated favorable biological properties, such as antioxidant activity, suppression of lipoxygenase (LOX)-1, cytotoxicity against cancer cells, and inhibition of the invasion and migration of cancer cells. This study further evaluated the effects of morusinol, a flavonoid derived from Morus alba root bark, on platelet aggregation and thromboxane B(2) (TXB(2) formation in vitro and thrombus formation in vivo. The antiplatelet potential of morusinol was measured using in vitro rabbit platelet aggregation and TXB(2) formation assays. Arterial thrombus formation was investigated using an in vivo ferric chloride (FeCl(3)-induced thrombosis model. Morusinol significantly inhibited collagen- and arachidonic acid-induced platelet aggregation and TXB(2) formation in cultured platelets in a concentration-dependent manner. Thrombus formation was reduced by 32.1, 42.0, and 99.0% for collagen-induced TXB(2) formation, and 8.0, 24.1, and 29.2% for arachadonic acid-induced TXB(2) formation, with 5, 10, and 30 µg/mL morusinol, respectively. Moreover, oral morusinol (20 mg/kg) or aspirin (20 mg/kg) for three days significantly increased the time to occlusion in vivo by 20.3±5.0 or 6.8±2.9 min, respectively, compared with the control (1% CMC, carboxymethyl cellulose). Taken together, these results indicate that morusinol may significantly inhibit arterial thrombosis in vivo due to antiplatelet activity. Thus, morusinol may exert beneficial effects on transient ischemic attacks or stroke via the modulation of platelet activation.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

2013-11-07

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

Sulfatides partition disabled-2 in response to platelet activation.

Directory of Open Access Journals (Sweden)

Karen E Drahos

Full Text Available BACKGROUND: Platelets contact each other at the site of vascular injury to stop bleeding. One negative regulator of platelet aggregation is Disabled-2 (Dab2, which is released to the extracellular surface upon platelet activation. Dab2 inhibits platelet aggregation through its phosphotyrosine-binding (PTB domain by competing with fibrinogen for alphaIIbbeta3 integrin receptor binding by an unknown mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Using protein-lipid overlay and liposome-binding assays, we identified that the N-terminal region of Dab2, including its PTB domain (N-PTB, specifically interacts with sulfatides. Moreover, we determined that such interaction is mediated by two conserved basic motifs with a dissociation constant (K(d of 0.6 microM as estimated by surface plasmon resonance (SPR analysis. In addition, liposome-binding assays combined with mass spectroscopy studies revealed that thrombin, a strong platelet agonist, cleaved N-PTB at a site located between the basic motifs, a region that becomes protected from thrombin cleavage when bound to sulfatides. Sulfatides on the platelet surface interact with coagulation proteins, playing a major role in haemostasis. Our results show that sulfatides recruit N-PTB to the platelet surface, sequestering it from integrin receptor binding during platelet activation. This is a transient recruitment that follows N-PTB internalization by an actin-dependent process. CONCLUSIONS/SIGNIFICANCE: Our experimental data support a model where two pools of Dab2 co-exist at the platelet surface, in both sulfatide- and integrin receptor-bound states, and their balance controls the extent of the clotting response.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance

Science.gov (United States)

Cuker, Adam; Husseinzadeh, Holleh; Lebedeva, Tatiana; Marturano, Joseph E.; Massefski, Walter; Lowery, Thomas J.; Lambert, Michele P.; Abrams, Charles S.; Weisel, John W.

2016-01-01

Objectives: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). Methods: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. Results: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. Conclusions: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests. PMID:28028118

Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

LENUS (Irish Health Repository)

Gegenbauer, Kristina

2013-11-01

Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

Endothelium depen dent factors of vasoconstriction (thromboxane B2 and vasodilation (6-prostaglandin F1α in children with primary arterial hype rten sion

Directory of Open Access Journals (Sweden)

Yu riy V. Marushko

2015-09-01

Full Text Available Background: Vasoconstrictor and vasodilator substances imbalance play a major role in the formation of arterial hypertension. But the ratio between thromboxane B2 and 6-prostaglandin F1α in children with various forms of primary arterial hypertension (PAH are insufficiently studied. Aim of the study: to explore the features of the content of thromboxane B2, 6-keto-PGF-1alfa and their correlation in children with different clinical and pathogenetic forms of PAH. Material and methods: The study involved 83 children aged 9 to 17 years. The first group included 32 children with stable PAH, the second – 32 children with labile PAH, the third (control group – 21 children with normal blood pressure. TXB2 and 6-PGF1α serum levels were investigated by ELISA. All children were held ambulatory blood pressure monitoring (ABPM. Results: Average TXB2 levels in boys were 25,05 ±6,43 ng/ml at stable PAH and 27,26 ±11,26 ng/ml at labile PAH, which exceeded their levels in the control group (p < 0,05. Girls’ TXB2 level was elevated at labile PAH (to 11,06 ±1,79 ng/ml, p < 0,05 and did not differ from the control group at stable PAH. Girls’ 6-PGF1α level was up to 3,41 ±0,52 ng/ml at stable PAH and up to 2,63 ±0,25 ng/ml at labile PAH. Conclusions: Violation of the ratio between endothelial vasoconstriction (thromboxane and vasodilatation (prostacyclin factors in boys with PAH is due to increased TXB2 levels compared with children with normal blood pressure (p < 0,05. Girls with PAH have better compensatory vasodilation opportunities compared with boys according to increased prostacyclin production. That prevents the progression of endothelial dysfunction and PAH stabilization in girls.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Science.gov (United States)

Semeraro, Fabrizio; Ammollo, Concetta T.; Morrissey, James H.; Dale, George L.; Friese, Paul; Esmon, Naomi L.

2011-01-01

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process. PMID:21673343

Diversity and impact of rare variants in genes encoding the platelet G protein-coupled receptors.

Science.gov (United States)

Jones, Matthew L; Norman, Jane E; Morgan, Neil V; Mundell, Stuart J; Lordkipanidzé, Marie; Lowe, Gillian C; Daly, Martina E; Simpson, Michael A; Drake, Sian; Watson, Steve P; Mumford, Andrew D

2015-04-01

Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70 % had global minor allele frequency (MAF) < 0.05 %. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21 %) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF< 1 % and 22 with MAF≥ 1 %). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.

Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer

OpenAIRE

O'Sullivan, Aine G.; Mulvaney, Eamon P.; Hyland, Paula B.; Kinsella, B. Therese

2015-01-01

The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TP? and TP? isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TP?/TP?-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenet...

Platelet size and age determine platelet function independently

International Nuclear Information System (INIS)

Thompson, C.B.; Jakubowski, J.A.; Quinn, P.G.; Deykin, D.; Valeri, C.R.

1984-01-01

A study was undertaken to examine the interaction of platelet size and age in determining in vitro platelet function. Baboon megakaryocytes were labeled in vivo by the injection of 75Se-methionine. Blood was collected when the label was predominantly associated with younger platelets (day 2) and with older platelets (day 9). Size-dependent platelet subpopulations were prepared on both days by counterflow centrifugation. The reactivity of each platelet subpopulation was determined on both days by measuring thrombin-induced aggregation. Platelets were fixed after partial aggregation had occurred by the addition of EDTA/formalin. After removal of the aggregated platelets by differential centrifugation, the supernatant medium was assayed for remaining platelets and 75Se radioactivity. Comparing day 2 and day 9, no significant difference was seen in the rate of aggregation of a given subpopulation. However, aggregation was more rapid in the larger platelet fractions than in the smaller ones on both days. A greater percentage of the 75Se radioactivity appeared in the platelet aggregates on day 2 than on day 9. This effect was independent of platelet size, as it occurred to a similar extent in the unfractionated platelets and in each of the size-dependent platelet subpopulations. The data indicate that young platelets are more active than older platelets. This study demonstrates that size and age are both determinants of platelet function, but by independent mechanisms

In situ microdialysis of intramuscular prostaglandin and thromboxane in contracting skeletal muscle in humans

DEFF Research Database (Denmark)

Karamouzis, M; Langberg, Henning; Skovgaard, D

2001-01-01

Arachidonic acid metabolites, especially prostacyclin I2, are regulators of vascular tone, and may be released from contracting muscle. In the present study, the influence of exercise on accumulation of prostaglandins and thromboxane in skeletal muscle was determined by the use of microdialysis...

Abnormal megakaryocyte development and platelet function in Nbeal2−/− mice

Science.gov (United States)

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

2013-01-01

Gray platelet syndrome (GPS) is an inherited bleeding disorder associated with macrothrombocytopenia and α-granule-deficient platelets. GPS has been linked to loss of function mutations in NEABL2 (neurobeachin-like 2), and we describe here a murine GPS model, the Nbeal2−/− mouse. As in GPS, Nbeal2−/− mice exhibit splenomegaly, macrothrombocytopenia, and a deficiency of platelet α-granules and their cargo, including von Willebrand factor (VWF), thrombospondin-1, and platelet factor 4. The platelet α-granule membrane protein P-selectin is expressed at 48% of wild-type levels and externalized upon platelet activation. The presence of P-selectin and normal levels of VPS33B and VPS16B in Nbeal2−/− platelets suggests that NBEAL2 acts independently of VPS33B/VPS16B at a later stage of α-granule biogenesis. Impaired Nbeal2−/− platelet function was shown by flow cytometry, platelet aggregometry, bleeding assays, and intravital imaging of laser-induced arterial thrombus formation. Microscopic analysis detected marked abnormalities in Nbeal2−/− bone marrow megakaryocytes, which when cultured showed delayed maturation, decreased survival, decreased ploidy, and developmental abnormalities, including abnormal extracellular distribution of VWF. Our results confirm that α-granule secretion plays a significant role in platelet function, and they also indicate that abnormal α-granule formation in Nbeal2−/− mice has deleterious effects on megakaryocyte survival, development, and platelet production. PMID:23861251

Platelet kinetics with indium-111 platelets: comparison with chromium-51 platelets

International Nuclear Information System (INIS)

Peters, A.M.; Lavender, J.P.

1983-01-01

The application of 111In-oxine to platelet labeling has contributed to the understanding of platelet kinetics along three lines: 1. It allows the measurement of new parameters of splenic function, such as the intrasplenic platelet transit time, which has shed new light on the physiology of splenic blood cell handling. 2. It facilitates the measurement of platelet life span in conditions, such as ITP, in which 51Cr may undergo undesirable elution from the platelet as a result of platelet-antibody interaction. 3. It allows the determination of the fate of platelets, that is, the site of platelet destruction in conditions in which reduced platelet life span is associated with abnormal platelet consumption, as a result of either premature destruction of ''abnormal'' platelets by the RE system, or the consumption (or destruction) of normal platelets after their interaction with an abnormal vasculature. Future research using 111In platelets may yield further valuable information on the control as well as the significance of intrasplenic platelet pooling, on the role of platelets in the development of chronic vascular lesions, and on the sites of platelet destruction in ITP. With regard to the latter, methods will have to be developed for harvesting sufficient platelets representative of the total circulating platelet population from severely thrombocytopenic patients for autologous platelet labeling. This would avoid the use of homologous platelets, which is likely to be responsible for some of the contradictory data relating to the use of radiolabeled platelet studies for the prediction of the response of patients with ITP to splenectomy

Valsartan decreases platelet activity and arterial thrombotic events in elderly patients with hypertension.

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Wu, Fang; Wang, Hong-Yan; Cai, Fan; Wang, Ling-Jie; Zhang, Feng-Ru; Chen, Xiao-Nan; Yang, Qian; Jiang, Meng-Hui; Wang, Xue-Feng; Shen, Wei-Feng

2015-01-20

Angiotensin type 1 receptor (AT 1 R) antagonists are extensively used for blood pressure control in elderly patients with hypertension. This study aimed to investigate the inhibitory effects of AT 1 R antagonist valsartan on platelet aggregation and the occurrence of cardio-cerebral thrombotic events in elderly patients with hypertension. Two-hundred and ten patients with hypertension and aged > 60 years were randomized to valsartan (n = 140) or amlodipine (n = 70) on admission. The primary endpoint was platelet aggregation rate (PAR) induced by arachidonic acid at discharge, and the secondary endpoint was the rate of thrombotic events including brain infarction and myocardial infarction during follow-up. Human aortic endothelial cells (HAECs) were stimulated by angiotensin II (Ang II, 100 nmol/L) with or without pretreatment of valsartan (100 nmol/L), and relative expression of cyclooxygenase-2 (COX-2) and thromboxane B 2 (TXB 2 ) and both p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kB (NF-kB) activities were assessed. Statistical analyses were performed by GraphPad Prism 5.0 software (GraphPad Software, Inc., California, USA). PAR was lower after treatment with valsartan (11.49 ± 0.69% vs. 18.71 ± 2.47%, P event rate in patients treated with valsartan (14.3% vs. 32.8%, P = 0.002). Relative expression of COX-2 and secretion of TXB 2 with concordant phosphorylation of p38MAPK and NF-kB were increased in HAECs when stimulated by Ang II (100 nmol/L) but were significantly decreased by valsartan pretreatment (100 nmol/L). AT 1 R antagonist valsartan decreases platelet activity by attenuating COX-2/TXA 2 expression through p38MAPK and NF-kB pathways and reduces the occurrence of cardio-cerebral thrombotic events in elderly patients with hypertension.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

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Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M; Kahr, Walter H A; Hartwig, John H; Falet, Hervé

2015-07-02

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. © 2015 by The American Society of Hematology.

A Novel Role of Eruca sativa Mill. (Rocket Extract: Antiplatelet (NF-κB Inhibition and Antithrombotic Activities

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Eduardo Fuentes

2014-12-01

Full Text Available Background: Epidemiological studies have shown the prevention of cardiovascular diseases through the regular consumption of vegetables. Eruca sativa Mill., commonly known as rocket, is a leafy vegetable that has anti-inflammatory activity. However, its antiplatelet and antithrombotic activities have not been described. Methods: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL, was evaluated on human platelets: (i P-selectin expression by flow cytometry; (ii platelet aggregation induced by ADP, collagen and arachidonic acid; (iii IL-1β, TGF-β1, CCL5 and thromboxane B2 release; and (iv activation of NF-κB and PKA by western blot. Furthermore, (v antithrombotic activity (200 mg/kg and (vi bleeding time in murine models were evaluated. Results: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL inhibited P-selectin expression and platelet aggregation induced by ADP. The release of platelet inflammatory mediators (IL-1β, TGF-β1, CCL5 and thromboxane B2 induced by ADP was inhibited by Eruca sativa Mill. aqueous extract. Furthermore, Eruca sativa Mill. aqueous extract inhibited NF-κB activation. Finally, in murine models, Eruca sativa Mill. aqueous extract showed significant antithrombotic activity and a slight effect on bleeding time. Conclusion: Eruca sativa Mill. presents antiplatelet and antithrombotic activity.

Platelet activation during preparation of platelet concentrates: a comparison of the platelet-rich plasma and the buffy coat methods

NARCIS (Netherlands)

Fijnheer, R.; Pietersz, R. N.; de Korte, D.; Gouwerok, C. W.; Dekker, W. J.; Reesink, H. W.; Roos, D.

1990-01-01

The activation of platelets during the preparation of platelet concentrates (PCs) by two methods was compared. To eliminate interdonor differences, 2 units of whole blood were pooled and subsequently divided into two batches. From one batch, the platelets were harvested as pelleted platelets from

Platelet indices and glucose control in type 1 and type 2 diabetes mellitus: A case-control study.

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Zaccardi, F; Rocca, B; Rizzi, A; Ciminello, A; Teofili, L; Ghirlanda, G; De Stefano, V; Pitocco, D

2017-10-01

The relationship between platelet indices and glucose control may differ in type 1 (T1DM) and type 2 (T2DM) diabetes. We aimed to investigate differences in mean platelet volume (MPV), platelet count, and platelet mass between patients with T1DM, T2DM, and healthy controls and to explore associations between these platelet indices and glucose control. A total of 691 T1DM and 459 T2DM patients and 943 control subjects (blood donors) were included. HbA1c was measured in all subjects with diabetes and 36 T1DM patients further underwent 24 h-continuous glucose monitoring to estimate short-term glucose control (glucose mean and standard deviation). Adjusting for age and sex, platelet count was higher and MPV lower in both T1DM and T2DM patients vs control subjects, while platelet mass (MPV × platelet count) resulted higher only in T2DM. Upon further adjustment for HbA1c, differences in platelet count and mass were respectively 19.5 × 10 9 /L (95%CI: 9.8-29.3; p 1) and 101 fL/nL (12-191; p = 0.027) comparing T2DM vs T1DM patients. MPV and platelet count were significantly and differently related in T2DM patients vs both T1DM and control subjects; this difference was maintained also accounting for HbA1c, age, and sex. Platelet mass and the volume-count relationship were significantly related to HbA1c only in T1DM patients. No associations were found between platelet indices and short-term glucose control. By accounting for confounders and glucose control, our data evidenced higher platelet mass and different volume-count kinetics in subjects with T2DM vs T1DM. Long-term glucose control seemed to influence platelet mass and the volume-count relationship only in T1DM subjects. These findings suggest different mechanisms behind platelet formation in T1DM and T2DM patients with long-term glycaemic control being more relevant in T1DM than T2DM. Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian

Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

International Nuclear Information System (INIS)

Blank, M.L.; Lee, T.; Cress, E.A.; Malone, B.; Fitzgerald, V.; Snyder, F.

1984-01-01

The metabolic pathway for 1-alkyl-2-acetyl-sn-glycerols, a recently discovered biologically active neutral lipid class, was elucidated in experiments conducted with rabbit platelets. The total lipid extract obtained from platelets incubated with 1-[1-,2- 3 H]alkyl-2-acetyl-sn-glycerols or 1-alkyl-2-[ 3 H]acetyl-sn-glycerols contained at least six metabolic products. The six metabolites, identified on the basis of chemical and enzymatic reactions combined with thin-layer or high-performance liquid chromatographic analyses, corresponded to 1-alkyl-sn-glycerols, 1-alkyl-2-acetyl-sn-glycero-3-phosphates, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholines, and 1-alkyl-2-actyl-sn-glycero-3-phosphocholines (platelet activating factor). These results indicate that the metabolic pathway for alkylacetylglycerols involves reaction steps catalyzed by the following enzymatic activities: choline- and ethanolamine- phosphotransferases, acetyl-hydrolase, an acyltransferase, and a phosphotransferase. The step responsible for the biosynthesis of platelet activating factor would appear to be the most important reaction in this pathway and this product could explain the hypotensive activities previously described for alkylacetyl-(or propionyl)-glycerols. Of particular interest was the preference exhibited for the utilization of the 1-hexadecyl-2-acetyl-sn-glycerol species in the formation of platelet activating factor

Platelet Counts in Insoluble Platelet-Rich Fibrin Clots: A Direct Method for Accurate Determination

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Yutaka Kitamura

2018-02-01

Full Text Available Platelet-rich fibrin (PRF clots have been used in regenerative dentistry most often, with the assumption that growth factor levels are concentrated in proportion to the platelet concentration. Platelet counts in PRF are generally determined indirectly by platelet counting in other liquid fractions. This study shows a method for direct estimation of platelet counts in PRF. To validate this method by determination of the recovery rate, whole-blood samples were obtained with an anticoagulant from healthy donors, and platelet-rich plasma (PRP fractions were clotted with CaCl2 by centrifugation and digested with tissue-plasminogen activator. Platelet counts were estimated before clotting and after digestion using an automatic hemocytometer. The method was then tested on PRF clots. The quality of platelets was examined by scanning electron microscopy and flow cytometry. In PRP-derived fibrin matrices, the recovery rate of platelets and white blood cells was 91.6 and 74.6%, respectively, after 24 h of digestion. In PRF clots associated with small and large red thrombi, platelet counts were 92.6 and 67.2% of the respective total platelet counts. These findings suggest that our direct method is sufficient for estimating the number of platelets trapped in an insoluble fibrin matrix and for determining that platelets are distributed in PRF clots and red thrombi roughly in proportion to their individual volumes. Therefore, we propose this direct digestion method for more accurate estimation of platelet counts in most types of platelet-enriched fibrin matrix.

Characterization of the stimulation of human platelets by stable analogues of PGH2/TXA2

International Nuclear Information System (INIS)

Morinelli, T.A.

1987-01-01

The specific effects of the TXA 2 /PGH 2 analogues, U46619 (9,11-dideoxy,9α-11α-methanoepoxy-PGF/sub 2α/), and 9,11 aza-PGH 2 , on human platelet shape change, myosin light chain phosphorylation, serotonin release, fibrinogen receptor exposure and platelet aggregation were measured and compared with binding of 3 H-U46619 to platelets. Shape change and myosin light chain phosphorylation were found to saturable and dose dependent. These two effects were competitively inhibited by specific antagonists of TXA 2 /PGH 2 receptors (BM13177 and I-PTA-OH) indicating that they are receptor mediated. Binding of 3 H-U46619 showed two components. Occupancy of high affinity binding sites correlated with platelet shape change and myosin and light chain phosphorylation. A second component with an apparent K/sub d/ of 1.46 +/- 0.47 μM, may represent a second, low-affinity site. Therefore, the platelet release reaction as not directly correlated with occupancy of high affinity receptors but could be related to the second binding component of U46619. Fibrinogen receptor exposure and platelet aggregation caused by U46619 appeared to be events mediated by the release of ADP from platelet dense granules

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

International Nuclear Information System (INIS)

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

1981-01-01

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

Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

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Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

2017-08-29

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to platelet hyperactivity and limits antiplatelet drug efficacy in type 2 diabetes mellitus. © 2017 American Heart Association, Inc.

Ristocetin induces phosphorylated-HSP27 (HSPB1) release from the platelets of type 2 DM patients: Anti-platelet agent-effect on the release.

Science.gov (United States)

Tokuda, Haruhiko; Kuroyanagi, Gen; Onuma, Takashi; Enomoto, Yukiko; Doi, Tomoaki; Iida, Hiroki; Otsuka, Takanobu; Ogura, Shinji; Iwama, Toru; Kojima, Kumi; Kozawa, Osamu

2018-04-01

It has been previously reported that HSP27 is released from platelets in type 2 diabetes mellitus (DM) patients according to phosphorylation. In the present study, we investigated the effect of ristocetin, a glycoprotein (GP)Ib/IX/V activator, on the release of HSP27 and the effect of anti-platelet agents, such as acetylsalicylic acid (ASA), on this release. Forty-six patients with type 2 DM were recruited, and classified into two groups depending on administration of anti-platelet agents, resulting in 31 patients without these agents (control group) and 15 patients with them (anti-platelet group). Ristocetin potently induced the aggregation of platelets in the two groups. Ristocetin-induced changes of the area under the curve of light transmittance and the ratio of the size of platelet aggregates in the anti-platelet group were slightly different from those in the control group. On the other hand, the levels of phosphorylated-HSP27 induced by ristocetin in the platelets from a patient of the anti-platelet group taking ASA were significantly lower than those from a patient of the control group. The levels of HSP27 release from the ristocetin-stimulated platelets were significantly correlated with the levels of phosphorylated-HSP27 in the platelets from patients in the two groups. The levels of HSP27 release and those of platelet-derived growth factor-AB (PDGF-AB) secretion stimulated by ristocetin in the anti-platelet group were lower than those in the control group. In addition, the levels of HSP27 release and those of PDGF-AB secretion stimulated by ADP in the anti-platelet group were lower than those in the control group. These results strongly suggest that anti-platelet agents inhibit the HSP27 release from platelets stimulated by ristocetin but not the aggregation in type 2 DM patients.

Renal cells activate the platelet receptor CLEC-2 through podoplanin

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Christou, Charita M.; Pearce, Andrew C.; Watson, Aleksandra A.; Mistry, Anita R.; Pollitt, Alice Y.; Fenton-May, Angharad E.; Johnson, Louise A.; Jackson, David G.; Watson, Steve P.; O'Callaghan, Chris A.

2009-01-01

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin, rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing human immunodeficiency virus type 1 (HIV-1). This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of this study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to 293T cells in which the HIV can be grown. Further, 293T cells activate both platelets and CLEC-2-transfected DT-40 B cells. The transmembrane protein podoplanin was identified on 293T cells and demonstrated to mediate both binding of 293T cells to CLEC-2 and 293T cell activation of CLEC-2-transfected DT-40 B cells. Podoplanin is expressed on renal cells (podocytes). Further, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5 ± 3.7μM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells. PMID:18215137

Stanniocalcin 2 Regulates Non-capacitative Ca2+ Entry and Aggregation in Mouse Platelets

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Esther López

2018-03-01

Full Text Available Stanniocalcin 2 (STC2 is a fish protein that controls body Ca2+ and phosphate metabolism. STC2 has also been described in mammals, and as platelet function highly depends on both extracellular and intracellular Ca2+, we have explored its expression and function in these cells. STC2−/− mice exhibit shorter tail bleeding time than WT mice. Platelets from STC2-deficient mice showed enhanced aggregation, as well as enhanced Ca2+ mobilization in response to the physiological agonist thrombin (Thr and the diacylglycerol analog, OAG, a selective activator of the non-capacitative Ca2+ entry channels. Interestingly, platelets from STC2−/− mice exhibit attenuated interaction between STIM1 and Orai1 in response to Thr, thus suggesting that STC2 is required for Thr-evoked STIM1-Orai1 interaction and the subsequent store-operated Ca2+ entry (SOCE. We have further assessed possible changes in the expression of the most relevant channels involved in non-capacitative Ca2+ entry in platelets. Then, protein expression of Orai3, TRPC3 and TRPC6 were evaluated by Western blotting, and the results revealed that while the expression of Orai3 was enhanced in the STC2-deficient mice, others like TRPC3 and TRPC6 remains almost unaltered. Summarizing, our results provide for the first time evidence for a role of STC2 in platelet physiology through the regulation of agonist-induced Ca2+ entry, which might be mediated by the regulation of Orai3 channel expression.

Enu mutagenesis identifies a novel platelet phenotype in a loss-of-function Jak2 allele.

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Nicole M Anderson

Full Text Available Utilizing ENU mutagenesis, we identified a mutant mouse with elevated platelets. Genetic mapping localized the mutation to an interval on chromosome 19 that encodes the Jak2 tyrosine kinase. We identified a A3056T mutation resulting in a premature stop codon within exon 19 of Jak2 (Jak2(K915X, resulting in a protein truncation and functionally inactive enzyme. This novel platelet phenotype was also observed in mice bearing a hemizygous targeted disruption of the Jak2 locus (Jak2(+/-. Timed pregnancy experiments revealed that Jak2(K915X/K915X and Jak2(-/- displayed embryonic lethality; however, Jak2(K915X/K915X embryos were viable an additional two days compared to Jak2(-/- embryos. Our data suggest that perturbing JAK2 activation may have unexpected consequences in elevation of platelet number and correspondingly, important implications for treatment of hematological disorders with constitutive Jak2 activity.

Influence of cytochrome 2C19 allelic variants on on-treatment platelet reactivity evaluated by five different platelet function tests.

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Gremmel, Thomas; Kopp, Christoph W; Moertl, Deddo; Seidinger, Daniela; Koppensteiner, Renate; Panzer, Simon; Mannhalter, Christine; Steiner, Sabine

2012-05-01

The antiplatelet effect of clopidogrel has been linked to cytochrome P450 2C19 (CYP2C19) carrier status. The presence of loss of function and gain of function variants were found to have a gene-dose effect on clopidogrel metabolism. However, genotyping is only one aspect of predicting response to clopidogrel and several platelet function tests are available to measure platelet response. Patients and methods We studied the influence of CYP2C19 allelic variants on on-treatment platelet reactivity as assessed by light transmission aggregometry (LTA), the VerifyNow P2Y12 assay, the VASP assay, multiple electrode aggregometry (MEA), and the Impact-R in 288 patients after stenting for cardiovascular disease. Allelic variants of CYP2C19 were determined with the Infiniti® CYP450 2C19+ assay and categorized into four metabolizer states (ultrarapid, extensive, intermediate, poor). Platelet reactivity increased linearly from ultrarapid to poor metabolizers using the VerifyNow P2Y12 assay (P = 0.04), the VASP assay (P = 0.02) and the Impact-R (P = 0.04). The proportion of patients with high on-treatment residual platelet reactivity (HRPR) identified by LTA, the VerifyNow P2Y12 assay and the VASP assay increased when the metabolizer status decreased, while no such relationship could be identified for results of MEA and Impact-R. The presence of loss of function variants (*2/*2, *2-8*/wt, *2/*17) was an independent predictor of HRPR in LTA and the VASP assay while it did not reach statistical significance in the VerifyNow P2Y12 assay, MEA, and the Impact-R. Depending on the type of platelet function test differences in the association of on-treatment platelet reactivity with CYP2C19 carrier status are observed. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quality assessment of platelet concentrates prepared by platelet rich plasma-platelet concentrate, buffy coat poor-platelet concentrate (BC-PC and apheresis-PC methods

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Singh Ravindra

2009-01-01

Full Text Available Background: Platelet rich plasma-platelet concentrate (PRP-PC, buffy coat poor-platelet concentrate (BC-PC, and apheresis-PC were prepared and their quality parameters were assessed. Study Design: In this study, the following platelet products were prepared: from random donor platelets (i platelet rich plasma - platelet concentrate (PRP-PC, and (ii buffy coat poor- platelet concentrate (BC-PC and (iii single donor platelets (apheresis-PC by different methods. Their quality was assessed using the following parameters: swirling, volume of the platelet concentrate, platelet count, WBC count and pH. Results: A total of 146 platelet concentrates (64 of PRP-PC, 62 of BC-PC and 20 of apheresis-PC were enrolled in this study. The mean volume of PRP-PC, BC-PC and apheresis-PC was 62.30±22.68 ml, 68.81±22.95 ml and 214.05±9.91 ml and ranged from 22-135 ml, 32-133 ml and 200-251 ml respectively. The mean platelet count of PRP-PC, BC-PC and apheresis-PC was 7.6±2.97 x 1010/unit, 7.3±2.98 x 1010/unit and 4.13±1.32 x 1011/unit and ranged from 3.2-16.2 x 1010/unit, 0.6-16.4 x 1010/unit and 1.22-8.9 x 1011/unit respectively. The mean WBC count in PRP-PC (n = 10, BC-PC (n = 10 and apheresis-PC (n = 6 units was 4.05±0.48 x 107/unit, 2.08±0.39 x 107/unit and 4.8±0.8 x 106/unit and ranged from 3.4 -4.77 x 107/unit, 1.6-2.7 x 107/unit and 3.2 - 5.2 x 106/unit respectively. A total of 26 units were analyzed for pH changes. Out of these units, 10 each were PRP-PC and BC-PC and 6 units were apheresis-PC. Their mean pH was 6.7±0.26 (mean±SD and ranged from 6.5 - 7.0 and no difference was observed among all three types of platelet concentrate. Conclusion: PRP-PC and BC-PC units were comparable in terms of swirling, platelet count per unit and pH. As expected, we found WBC contamination to be less in BC-PC than PRP-PC units. Variation in volume was more in BC-PC than PRP-PC units and this suggests that further standardization is required for preparation of BC

LDL oxidation by platelets propagates platelet activation via an oxidative stress-mediated mechanism.

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Carnevale, Roberto; Bartimoccia, Simona; Nocella, Cristina; Di Santo, Serena; Loffredo, Lorenzo; Illuminati, Giulio; Lombardi, Elisabetta; Boz, Valentina; Del Ben, Maria; De Marco, Luigi; Pignatelli, Pasquale; Violi, Francesco

2014-11-01

Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation. Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency. Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists. Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

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Ting-Lin Yen

2014-01-01

Full Text Available 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 (PLCγ2, 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.

Haplotype of platelet receptor P2RY12 gene is associated with residual clopidogrel on-treatment platelet reactivity.

Science.gov (United States)

Nie, Xiao-Yan; Li, Jun-Lei; Zhang, Yong; Xu, Yang; Yang, Xue-Li; Fu, Yu; Liang, Guang-Kai; Lu, Yun; Liu, Jian; Shi, Lu-Wen

To investigate a possible association between common variations of the P2RY12 and the residual clopidogrel on-treatment platelet reactivity after adjusting for the influence of CYP2C19 tested by thromboelastography (TEG). One hundred and eighty patients with acute coronary syndrome (ACS) treated with clopidogrel and aspirin were included and platelet function was assessed by TEG. Five selected P2RY12 single nucleotide polymorphisms (SNPs; rs6798347, rs6787801, rs6801273, rs6785930, and rs2046934), which cover the common variations in the P2RY12 gene and its regulatory regions, and three CYP2C19 SNPs ( * 2, * 3, * 17) were genotyped and possible haplotypes were analyzed. The high on-treatment platelet reactivity (HTPR) prevalence defined by a platelet inhibition rate <30% by TEG in adenosine diphosphate (ADP)-channel was 69 (38.33%). Six common haplotypes were inferred from four of the selected P2RY12 SNPs (denoted H 0 to H 5 ) according to the linkage disequilibrium R square (except for rs2046934). Haplotype H 1 showed a significantly lower incidence of HTPR than the reference haplotype (H 0 ) in the total study population while haplotypes H 1 and H 2 showed significantly lower incidences of HTPR than H 0 in the nonsmoker subgroup after adjusting for CYP2C19 effects and demographic characteristics. rs2046934 (T744C) did not show any significant association with HTPR. The combination of common P2RY12 variations including regulatory regions rather than rs2046934 (T744C) that related to pharmacodynamics of clopidogrel in patients with ACS was independently associated with residual on-clopidogrel platelet reactivity. This is apart from the established association of the CYP2C19. This association seemed more important in the subgroup defined by smoking.

Molecular mechanisms of platelet P2Y(12) receptor regulation.

Science.gov (United States)

Cunningham, Margaret R; Nisar, Shaista P; Mundell, Stuart J

2013-02-01

Platelets are critical for haemostasis, however inappropriate activation can lead to the development of arterial thrombosis, which can result in heart attack and stroke. ADP is a key platelet agonist that exerts its actions via stimulation of two surface GPCRs (G-protein-coupled receptors), P2Y(1) and P2Y(12). Similar to most GPCRs, P2Y receptor activity is tightly regulated by a number of complex mechanisms including receptor desensitization, internalization and recycling. In the present article, we review the molecular mechanisms that underlie P2Y(1) and P2Y(12) receptor regulation, with particular emphasis on the structural motifs within the P2Y(12) receptor, which are required to maintain regulatory protein interaction. The implications of these findings for platelet responsiveness are also discussed.

Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

International Nuclear Information System (INIS)

Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H.

1990-01-01

The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[ 3 H]ethylcarboxamidoadenosine [( 3 H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [ 3 H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors

Comparison of Modified Impedance Whole Blood Platelet Aggregation Method Detecting Platelet Function in ACS Patients with Different CYP2C19 Genotypes.

Science.gov (United States)

Cui, Chanjuan; Qiao, Rui; Zhang, Jie

2016-01-01

A reliable laboratory test to monitor onclopidogrel platelet reactivity (PR) is very necessary. In addition, genetic factors also play an important part in onclopidogrel PR. This study aimed to modify the original impedance whole blood platelet aggregation assay associated with the release assay to monitor onclopidogrel PR and assess their relationship with genotype. We adjusted the concentration of calcium in the in vitro reaction system of platelet aggregation to modify the original impedance whole blood platelet aggregation assay. Meanwhile, chronolume, which quantified the adenosine triphosphate (ATP) released from platelet dense granules, is added to this reaction system to reflect the platelet release function. In the modified assay, platelet magnified activation time (MAT) and the maximal platelet ATP release value (RV) were used to reflect platelet function parameters. In the original assay, the electrical resistance (omega) and RV were used to reflect platelet function parameters. Onclopidogrel PR was detected by the original impedance whole blood platelet aggregation assay, modified assay, and flow cytometric vasodilator stimulated phosphoprotein (VASP) assay in 168 patients with acute coronary syndromes (ACS). CYP2C19*2 and CYP2C19*3 polymorphisms were also detected in all of these patients. This modified method showed that when 12.5 microL CaCl2 (0.2 mmol/L) was added to the reaction system, MAT was appropriate (93 +/- 23 seconds). The CVs for the modified impedance assay and release assay were 9.31% and 6.13%, respectively. The mean VASP-PRI in the patient group treated with clopidogrel was significantly lower than that in the control group without antiplatelet therapy (54.88 +/- 16.81% vs. 79.86 +/- 10.24%, p 50% group were shorter than that in the PRI 50% group were higher than that in the PRI omega) and RV of the original method showed no differences between the two groups [0 (0-2) vs. 0 (0-1.25), 0.05 (0-0.25) vs. 0.08 (0-0.24); p > 0.05, p > 0

Salvia miltiorrhiza Bunge (Danshen) extract attenuates permanent cerebral ischemia through inhibiting platelet activation in rats.

Science.gov (United States)

Fei, Yu-Xiang; Wang, Si-Qi; Yang, Li-Jian; Qiu, Yan-Ying; Li, Yi-Ze; Liu, Wen-Yuan; Xi, Tao; Fang, Wei-Rong; Li, Yun-Man

2017-07-31

Danshen is a crude herbal drug isolated from dried roots of Salvia miltiorrhiza Bunge. This plant is widely used in oriental medicine for the treatment of cardiovascular and cerebrovascular diseases. The supercritical CO 2 extract from Danshen (SCED) (57.85%, 5.67% and 4.55% for tanshinone IIA, tanshinone I and cryptotanshinone respectively) was studied in this article, whose potential molecular mechanism remains unclear, especially in anti-thrombosis. The present study was designed to observe the protective effect of SCED on ischemic stroke in rats and to explore the underlying anti-thrombosis mechanism. Following induction of cerebral ischemia in rats by permanent middle cerebral artery occlusion (pMCAO). Neurological defect score, cerebral blood flow, infarct size, and brain edema were measured to evaluate the injury. Arteriovenous shunt thrombosis model and adenosine 5'-diphosphate (ADP) induced acute pulmonary embolism model were conducted to estimate the antithrombotic effect of SCED. In order to investigate the effects of SCED on platelet aggregation, rat platelet-rich-plasma (PRP) were incubated with SCED prior to the addition of the stimuli (ADP or 9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α (U46619)). Aggregation was monitored in a light transmission aggregometer. Inhibitory effect of SCED on thromboxane A2 (TXA 2 ) release was detected by ELISA kit. Phospholipase C (PLC)/ Protein kinase C (PKC) signaling pathway was analyzed by a Western blot technique. The effect of the SCED was also studied in vivo on bleeding time in mice. SCED improved the neurological defect score, increased cerebral blood flow, reduced infarct size and alleviated brain edema in rats exposed to pMCAO. After administration of SCED, thrombosis formation in arteriovenous shunt was inhibited and recovery time in pulmonary embolism was shortened. The inhibitory effect of SCED on platelet activation was further confirmed by TXB 2 ELISA kit and Western blot analysis of PLC

Effect of preoperative antiplatelet drugs on vascular prostacyclin synthesis.

Science.gov (United States)

Karwande, S V; Weksler, B B; Gay, W A; Subramanian, V A

1987-03-01

Patients undergoing aortocoronary bypass using autogenous saphenous veins were randomly divided into three comparable groups. Group 1 (n = 10) acted as a control, Group 2 (n = 14) received 80 mg of aspirin at midnight before the operation, and Group 3 (n = 12) received 80 mg of aspirin and 75 mg of dipyridamole at midnight and an additional 75-mg dose of dipyridamole at 6 AM. The purpose was to determine which regimen would maximally inhibit platelet function without depressing vascular prostacyclin synthesis. Serum thromboxane A2, saphenous vein wall and aortic wall prostacyclin, platelet aggregation, and bleeding time were measured in all patients. None was restarted on a regimen of aspirin or dipyridamole postoperatively. Aspirin alone and in combination with dipyridamole significantly inhibited thromboxane A2 and platelet aggregation in all treated patients but spared venous prostacyclin synthesis. Aortic prostacyclin synthesis was partially inhibited in both treated groups. Chest tube drainage was comparable in all three groups. These results indicate that the combination of aspirin and dipyridamole offers no measurable advantage over aspirin alone in the perioperative period.

Platelet size does not correlate with platelet age.

Science.gov (United States)

Thompson, C B; Love, D G; Quinn, P G; Valeri, C R

1983-08-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.

Platelet size does not correlate with platelet age

International Nuclear Information System (INIS)

Thompson, C.B.; Love, D.G.; Quinn, P.G.; Valeri, C.R.

1983-01-01

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense-body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75 Se-methionine, (2) in vitro labeling with 51 Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75 Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51 Cr and 14 C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation

Single prostacyclin receptor of gel-filtered platelets provides a correlation with antiaggregatory potency of PGI2 mimics

International Nuclear Information System (INIS)

Eggerman, T.L.; Hartzell, C.J.; Selfe, S.; Andersen, N.H.

1987-01-01

Gel-filtered human platelets (GFP) display only a single binding site for [ 3 H]-PGI2: KD = 61nM, 234 fmol/10(8) platelets (1410 sites/platelet). Platelet-rich plasma (PRP) displays the same receptor density but the KD value increases to 123 nM due to protein binding of PGI2 which lowers its effective concentration. The [ 3 H]-PGI2/GFP binding assay has been used to evaluate the molecular basis of aggregation inhibition for prostacyclin analogs and mimics, three PGE type structures, and PGD2. Antiaggregatory IC50s and radioligand binding IC50s correlate for PGE2, E1, and six PGI2 analogs. PGD2, and to a lesser extent 6-oxo-PGE1, display greater antiaggregatory potency than expected based on PGI2-binding site affinity data

Evaluation of platelet aggregation in platelet concentrates: storage implications

Directory of Open Access Journals (Sweden)

Neiva Teresinha J.C.

2003-01-01

Full Text Available The use of hemo-derivatives is nowadays a fundamentally important therapeutic modality in the exercise of medicine. Among the various hemo-components employed, we have the platelet concentrate (PC, indicated in cases of hemorrhagic disturbances. We previously showed that platelet function in blood donors is reduced in their screening phase and after the separation process of PCs. Currently, we are providing evidence for the existence of biochemical and functional changes in PC preparations stored for three days at temperatures of 20 ± 2 ºC. Platelet concentrates from 40 healthy donors, collected in CPD anticoagulant and PL-146 polyvinylchloride containers, were examined in order to determine the pH value, pCO2 ,pO2 and lactate concentrations. In addition, the aggregation of platelets with thrombin and collagen were examined to evaluate platelet function. A pH increase from 7.07 ± 0.04 to 7.36 ± 0.07 (p < 0.01 was observed. The pCO2 concentration decreased progressively from 69.2 ± 7.7 mmHg to 28.8 ± 6.2 mmHg (p < 0.001 during the storage period. In contrast, pO2 value increase from 103.4 ± 30.6 to 152.3 ± 24.6 mmHg (p < 0.001 was evidenced during the 48 hours of storage. The lactate concentration increased from 17.97 ± 5.2 to 57.21 ± 5.7 mg/dl (p < 0.001. Platelet aggregation using 0.25 U/ml-thrombin and 2.0 µg/ml-collagen showed significant hypofunction from 61.8 ± 2.7% to 24.8 ± 9.8% and 62.7±5.0 to 33.4± 6.2 (p < 0.001, respectively. We concluded that the evaluated biochemical parameters and the platelet function changed significantly when the platelets were kept under routine storage conditions.

Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

International Nuclear Information System (INIS)

Kornecki, E.; Ehrlich, Y.H.; Egbring, R.; Gramse, M.; Seitz, R.; Eckardt, A.; Lukasiewicz, H.; Niewiarowski, S.

1988-01-01

The authors have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125 I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase. Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen-binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125 I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. They conclude that (1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and (2) human granulocyte elastase exposes active fibrongen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

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Monique Ramos de Oliveira Trugilho

2017-05-01

Full Text Available Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet

Differences in levels of platelet-derived microparticles in platelet components prepared using the platelet rich plasma, buffy coat, and apheresis procedures.

Science.gov (United States)

Noulsri, Egarit; Udomwinijsilp, Prapaporn; Lerdwana, Surada; Chongkolwatana, Viroje; Permpikul, Parichart

2017-04-01

There has been an increased interest in platelet-derived microparticles (PMPs) in transfusion medicine. Little is known about PMP status during the preparation of platelet concentrates for transfusion. The aim of this study is to compare the PMP levels in platelet components prepared using the buffy coat (BC), platelet-rich plasma platelet concentrate (PRP-PC), and apheresis (AP) processes. Platelet components were prepared using the PRP-PC and BC processes. Apheresis platelets were prepared using the Trima Accel and Amicus instruments. The samples were incubated with annexin A5-FITC, CD41-PE, and CD62P-APC. At day 1 after processing, the PMPs and activated platelets were determined using flow cytometry. Both the percentage and number of PMPs were higher in platelet components prepared using the Amicus instrument (2.6±1.8, 32802±19036 particles/μL) than in platelet components prepared using the Trima Accel instrument (0.5±0.4, 7568±5298 particles/μL), BC (1.2±0.6, 12,920±6426 particles/μL), and PRP-PC (0.9±0.6, 10731±5514 particles/μL). Both the percentage and number of activated platelets were higher in platelet components prepared using the Amicus instrument (33.2±13.9, 427553±196965 cells/μL) than in platelet components prepared using the Trima Accel instrument (16.2±6.1, 211209±87706 cells/μL), BC (12.9±3.2, 140624±41003 cells/μL), and PRP-PC (21.1±6.3, 265210±86257 cells/μL). The study suggests high variability of PMPs and activated platelets in platelet components prepared using different processes. This result may be important in validating the instruments involved in platelet blood collection and processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Rac1 is essential for phospholipase C-gamma2 activation in platelets

DEFF Research Database (Denmark)

Pleines, Irina; Elvers, Margitta; Strehl, Amrei

2008-01-01

isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC......Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta...... regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation...

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

Science.gov (United States)

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Ativação plaquetária na esclerose sistêmica e alternativas metodológicas para sua avaliação Platelet activation in systemic sclerosis and methodological options to its assessment

Directory of Open Access Journals (Sweden)

Paulo Sergio Massabki

2004-02-01

immunologic abnormalities. The microangiopathic aspect is responsible for the most severe and life-threatening features of the disease. The interaction between endothelium and platelets plays an important role in the pathophysiology of SSc. Evidence of platelet activation in SSc includes high plasma levels of platelet-derived substances (Von Willenbrand factor, platelet factor 4, thromboxane A2, and ß-thromboglobulin, circulating platelet aggregates, ultra structural abnormalities in platelets, and the presence of platelets adhered to the endothelium. This review addresses the principles and possible pitfalls of the main methods for evaluation of platelet activation and function. The assessment of the ability of platelet aggregation is performed with and without the addition of agonists (adenosine diphosphate, collagen, adrenaline. Platelet activation may be assessed by two ways: by measurement of plasma concentration of substances that are released as the platelets are activated (e.g., thromboxane, platelet factor 4 and by the measurement of membrane expression of molecules that are transported to the platelet membrane during the activation process (e.g., GMP-140, glycoprotein IIb/IIIa. A recent contribution to this field was the demonstration that neuron-specific enolase (NSE is released from the platelets into the blood in patients with active SSc. NSE, which is readily available in clinical laboratories with emphasis in tumor markers, may become a useful platelet activation marker in a series of clinical conditions.

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

Science.gov (United States)

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.

Inhibitory Effect of Flavonolignans on the P2Y12 Pathway in Blood Platelets.

Science.gov (United States)

Bijak, Michal; Szelenberger, Rafal; Dziedzic, Angela; Saluk-Bijak, Joanna

2018-02-10

Adenosine diphosphate (ADP) is the major platelet agonist, which is important in the shape changes, stability, and growth of the thrombus. Platelet activation by ADP is associated with the G protein-coupled receptors P2Y1 and P2Y12. The pharmacologic blockade of the P2Y12 receptor significantly reduces the risk of peripheral artery disease, myocardial infarction, ischemic stroke, and vascular death. Recent studies demonstrated the inhibition of ADP-induced blood platelet activation by three major compounds of the flavonolignans group: silybin, silychristin, and silydianin. For this reason, the aim of the current work was to verify the effects of silybin, silychristin, and silydianin on ADP-induced physiological platelets responses, as well as mechanisms of P2Y12-dependent intracellular signal transduction. We evaluated the effect of tested flavonolignans on ADP-induced blood platelets' aggregation in platelet-rich plasma (PRP) (using light transmission aggregometry), adhesion to fibrinogen (using the static method), and the secretion of PF-4 (using the ELISA method). Additionally, using the double labeled flow cytometry method, we estimated platelet vasodilator-stimulated phosphoprotein (VASP) phosphorylation. We demonstrated a dose-dependent reduction of blood platelets' ability to perform ADP-induced aggregation, adhere to fibrinogen, and secrete PF-4 in samples treated with flavonolignans. Additionally, we observed that all of the tested flavonolignans were able to increase VASP phosphorylation in blood platelets samples, which is correlated with P2Y12 receptor inhibition. All of these analyses show that silychristin and silybin have the strongest inhibitory effect on blood platelet activation by ADP, while silydianin also inhibits the ADP pathway, but to a lesser extent. The results obtained in this study clearly demonstrate that silybin, silychristin, and silydianin have inhibitory properties against the P2Y12 receptor and block ADP-induced blood platelet

Some aspects of rat platelet and serum phospholipase A2 activities

NARCIS (Netherlands)

Aarsman, A.J.; Roosenboom, C.F.P.; Geffen, G.E.W. van; Bosch, H. van den

1985-01-01

Rat platelet lysate contained appreciable phospholipase A2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A2 activity

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

Science.gov (United States)

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

2014-01-01

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

Effect of Continuous Positive Airway Pressure Ventilation on Platelet-activating Factor and Blood Coagulation Function in Patients with Obstructive Sleep Apnea-hypopnea Syndrome

International Nuclear Information System (INIS)

Chen Xiangkun; Sheng Chunyong

2010-01-01

To investigate the effect of continuous positive airway pressure ventilation (CPAP) on platelet-activating factor (PAF) expression and blood coagulation function in patients with obstructive sleep apnea-hypopnea syndrome (OSAS), the blood sample of 40 patients with OSAS were taken before treatment and on the day 30 after treatment respectively. PAF, thromboxane B 2 (TXB2), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrin(FIB) in patients and 37 health controls were detected. The results showed that PAF, TXB2, FIB in OSAS patients before treatment were significantly higher than those of after treatment and control group (P 0.05). There were abnormal expression of PAF and hypercoagulability in OSAS patients. CPAP could effectively decrease the expression of PAF, TXB 2 and could also correct dysfunction of blood coagulation. It had certain effect in lightening the clinical symptoms in OSAS patients. (authors)

Platelet oxidative stress and its relationship with cardiovascular diseases in type 2 diabetes mellitus patients.

Science.gov (United States)

El Haouari, Mohammed

2017-10-05

Enhanced platelet activation and thrombosis are linked to various cardiovascular diseases. Among other mechanisms, oxidative stress seems to play a pivotal role in platelet hyperactivity. Indeed, upon stimulation by physiological agonists, human platelets generate and release several types of reactive oxygen species (ROS) such as O2-, H2O2 or OH- , further amplifying the platelet activation response via various signalling pathways, including, formation of isoprostanes, Ca2+ mobilization and NO inactivation. Furthermore, excessive platelet ROS generation, incorporation of free radicals from environment and/or depletion of antioxidants induce pro-oxidant, pro-inflammatory and platelet hyperaggregability effects, leading to the incidence of cardiovascular events. Here, we review the current knowledge regarding the effect of oxidative stress on platelet signaling pathways and its implication in CVD such as type 2 diabetes mellitus. We also summarize the role of natural antioxidants included in vegetables, fruits and medicinal herbs in reducing platelet function via an oxidative stress-mediated mechanism. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Relationship between platelet and urinary 8-Iso-PGF2α levels in subjects with different degrees of NOX2 regulation.

Science.gov (United States)

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-06-14

Urinary 8-iso-PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8-iso-PGF2α contribute to urinary 8-iso-PGF2α. In a cross-sectional study, platelet, urinary, and serum 8-iso-PGF2α were determined in subjects with downregulation (X-linked chronic granulomatous disease [X-CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8-iso-PGF2α. Compared with controls, X-CGD patients had lower platelet, serum, and urinary 8-iso-PGF2α values; conversely, diabetic patients had higher values of 8-iso-PGF2α compared with controls. Urinary 8-iso-PGF2α significantly correlated with both platelet and serum 8-iso-PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8-iso-PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8-iso-PGF2α and that only 13% of it is of extravascular origin. The study suggests that NOX2 contributes to the formation of 8-iso-PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8-iso-PGF2α suggests that, at least partly, urinary 8-iso-PGF2α reflects platelet 8-iso-PGF2α production. Analysis of serum 8-iso-PGF2α may represent a novel tool to investigate the production of 8-iso-PGF2α by blood cells including platelets. URL: ClinicalTrials.gov. Unique Identifier: NCT01250340.

Study on the correlative analysis between the changes of immunofunction status of peripheral blood red blood cells and plasma thromboxane B2 (TXB2) level in patients with pregnancy induced hypertension (PIH)

International Nuclear Information System (INIS)

Liu Ailing; Zhou Dongxia; Wang Yuanyuan

2011-01-01

Objective: To explore the relationship between changes of immunofuction status of peripheral blood red blood cells and plasma thromboxane B 2 (TXB 2 ) level in patients with PIH. Methods: RBC immunofunction status was studied with immunologic methods and plasma TXB 2 Level was measured with RIA in 33 patients with PIH and 35 normal controls.Results Level of RBC-C3bRR was significantly lower and level of plasma TXB 2 was markedly higher in the patients than those in controls (P 2 (r=0.4084, P 2 levels in patients with PIH. (authors)

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

International Nuclear Information System (INIS)

Laduca, F.M.; Bell, W.R.; Bettigole, R.E.

1987-01-01

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 [ 3 H]serotonin, or alter the dose-responsive binding of 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

Low multiple electrode aggregometry platelet responses are not associated with non-synonymous variants in G-protein coupled receptor genes.

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Norman, Jane E; Lee, Kurtis R; Walker, Mary E; Murden, Sherina L; Harris, Jessica; Mundell, Stuart; J Murphy, Gavin; Mumford, Andrew D

2015-10-01

Multiple electrode aggregometry (MEA) improves prediction of thrombosis and bleeding in cardiac patients. However, the causes of inter-individual variation in MEA results are incompletely understood. We explore whether low MEA results are associated with platelet G-protein coupled receptor (GPCR) gene variants. The effects of P2Y12 receptor (P2Y12), thromboxane A2 receptor (TPα) and protease-activated receptor 1 (PAR1) dysfunction on the MEA ADP-test, ASPI-test and TRAP-test were determined using receptor antagonists. Cardiac surgery patients with pre-operative MEA results suggesting GPCR dysfunction were selected for P2Y12 (P2RY12), TPα (TBXA2R) and PAR1 (F2R) sequencing. In control blood samples, P2Y12, TPα or PAR1 antagonists markedly reduced ADP-test, ASPI-test and TRAP-test results respectively. In the 636 patients from a cohort of 2388 cardiac surgery patients who were not receiving aspirin or a P2Y12 blocker, the median ADP-test result was 75.1 U (range 4.8-153.2), ASPI-test 83.7 U (1.4-157.3) and TRAP-test 117.7 U (2.4-194.1), indicating a broad range of results unexplained by anti-platelet drugs. In 238 consenting patients with unexplained low MEA results, three P2RY12 variants occurred in 70/107 (65%) with suspected P2Y12 dysfunction and four TBXA2R variants occurred in 19/22 (86%) with suspected TPα dysfunction although the later group was too small to draw meaningful conclusions about variant frequency. All the variants were synonymous and unlikely to cause GPCR dysfunction. There were no F2R variants in the 109 cases with suspected PAR1 dysfunction. MEA results suggesting isolated platelet GPCR dysfunction were common in cardiac surgery patients, but were not associated with non-synonymous variants in P2RY12 or F2R. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

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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.

Heterogeneity of rabbit platelets

International Nuclear Information System (INIS)

Karpatkin, S.

1978-01-01

Rabbits were injected intravenously with a cohort platelet label, 75 Se-selenomethionine. Platelet-rich plasma was separated into five different platelet density fractions on each of seven days by repetitively centrifuging the same sample of platelet-rich plasma at increasing gravitational force. The heaviest platelet sediment fraction was enriched with larger platelets. The lightest platelet sediment fraction was enriched with smaller platelets. Incorporation of isotope into the heaviest platelet fraction was considerably greater than incorporation into the lightest platelet fraction. The mean platelet survival of the lightest two fractions was significantly shorter than that of the heaviest three fractions. SDS-polyacrylamide gel electrophoresis of the platelet cell sap generally revealed 10 prominent protein bands for the heaviest platelet fractions. The lightest platelet fraction had six absent to markedly diminished platelet proteins. The data are compatible with two models, (1) heavy-large platelets are, on average, young platelets which become lighter-smaller platelets while losing platelet membranes and cell sap components with time. (2) Heavy-large platelets and light-small platelets are produced independently by specific megakarocytes. The heavy-large platelets incorporate more isotope that lighter-smaller platelets (possibly because of their megakarocyte precursor). However, they are released earlier into the circulation than lighter-smaller platelets and are therefore younger platelets. The light-smaller platelets which are released later into the circulation have a shorter survival. (author)

Platelet-rich fibrin: Evolution of a second-generation platelet concentrate

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Sunitha Raja V

2008-01-01

Full Text Available Platelet-rich plasma (PRP is a platelet concentrate that has been used widely to accelerate soft-tissue and hard-tissue healing. The preparation of PRP has been described by several authors. Platelet-rich fibrin (PRF was first described by Choukroun et al. in France. It has been referred to as a second-generation platelet concentrate, which has been shown to have several advantages over traditionally prepared PRP. Its chief advantages include ease of preparation and lack of biochemical handling of blood, which makes this preparation strictly autologous. This article describes the evolution of this novel platelet concentrate, referred to as PRF.

NP-184[2-(5-methyl-2-furyl) benzimidazole], a novel orally active antithrombotic agent with dual antiplatelet and anticoagulant activities.

Science.gov (United States)

Kuo, Heng-Lan; Lien, Jin-Cherng; Chung, Ching-Hu; Chang, Chien-Hsin; Lo, Shyh-Chyi; Tsai, I-Chun; Peng, Hui-Chin; Kuo, Sheng-Chu; Huang, Tur-Fu

2010-06-01

The established antiplatelet and anticoagulant agents show beneficial effects in the treatment of thromboembolic diseases; however, these drugs still have considerable limitations. The effects of NP-184, a synthetic compound, on platelet functions, plasma coagulant activity, and mesenteric venule thrombosis in mice were investigated. NP-184 concentration-dependently inhibited the human platelet aggregation induced by collagen, arachidonic acid (AA), and U46619, a thromboxane (TX)A(2) mimic, with IC(50) values of 4.5 +/- 0.2, 3.9 +/- 0.1, and 9.3 +/- 0.5 microM, respectively. Moreover, NP-184 concentration-dependently suppressed TXA(2) formations caused by collagen and AA. In exploring effects of NP-184 on enzymes involved in TXA(2) synthesis, we found that NP-184 selectively inhibited TXA(2) synthase activity with an IC(50) value of 4.3 +/- 0.2 microM. Furthermore, NP-184 produced a right shift of the concentration-response curve of U46619, indicating a competitive antagonism on TXA(2)/prostaglandin H(2) receptor. Intriguingly, NP-184 also caused a concentration-dependent prolongation of the activated partial thromboplastin time (aPTT) with no changes in the prothrombin and thrombin time, indicating that it selectively impairs the intrinsic coagulation pathway. Oral administration of NP-184 significantly inhibited thrombus formation of the irradiated mesenteric venules in fluorescein sodium-treated mice without affecting the bleeding time induced by tail transection. However, after oral administration, NP-184 inhibited the ex vivo mouse platelet aggregation triggered by collagen and U46619 and also prolonged aPTT. Taken together, the dual antiplatelet and anticoagulant activities of NP-184 may have therapeutic potential as an oral antithrombotic agent in the treatment of thromboembolic disorders.

Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+

International Nuclear Information System (INIS)

Winters, K.J.; Eisenberg, P.R.; Jaffe, A.S.; Santoro, S.A.

1990-01-01

The effects of activation of plasminogen by streptokinase and tissue-type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125 I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca 2+ , conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca 2+ . The binding of activation-specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation

Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation

DEFF Research Database (Denmark)

Dahl, Sandra Rinne; Kleiveland, Charlotte Ramstad; Kassem, Moustapha

2009-01-01

An on-line strong cation-exchange (SCX)-reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B(2), TXB(3), leukotriene (LT) B(4), LTD(4) and lipoxin (LX) A(4) in cell...

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

Science.gov (United States)

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Assessment of quality of platelets preserved in plasma and platelet additive solution: A Malaysian experience

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Munirah Binti Mokhtar

2016-01-01

Full Text Available Background: A use of platelet additives solution (PAS improves storage conditions so as to give increased shelf life to platelets and to maintain hemostatic function. Objective: The present study was aimed to compare in vitro quality of platelet rich plasma (PRP-derived platelet concentrate (PC during extended period of storage in plasma and in additive solution (Composol PS and Fresenius. Study Design: Randomized 19 PCs each were used in the study for plasma and PAS as the storage medium. The measurement parameters, including pH, total white blood cell (WBC count, total platelet count, and platelet activation rate, were studied on day 1, day 5, and day 8 of the storage period. The sterility test was carried out on the eighth day of storage. Results: pH of PC suspended in PAS was significantly lower as compared to that in plasma (P < 0.001 for all the three days of sampling. The WBC count, both in plasma and in PAS, showed an acceptable values of being <0.2 Χ 10 9 /unit during the storage period. Platelet count in PAS was higher as compared to that in plasma, though it was not statistically significant. While both the groups showed increased platelet activation rate during the storage, the PCs suspended in PAS showed significantly higher platelet activation rate (p0.001. Results from sterility test showed no bacterial growth in the PCs in both the groups. Conclusion: Most parameters studied on platelet storage in suspending medium of native plasma and PAS remained well within the acceptable limits. However, the pH values and platelet activation rate significantly differed in PAS as compared with plasma.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

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Schweigel, Hardy; Geiger, Jörg; Beck, Florian; Buhs, Sophia; Gerull, Helwe; Walter, Ulrich; Sickmann, Albert; Nollau, Peter

2013-03-01

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

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Fediuk, J; Sikarwar, A S; Lizotte, P P; Hinton, M; Nolette, N; Dakshinamurti, S

2015-02-01

Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles.

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Rank, A; Nieuwland, R; Liebhardt, S; Iberer, M; Grützner, S; Toth, B; Pihusch, R

2011-02-01

Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice

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Tomberg, Kärt; Khoriaty, Rami; Westrick, Randal J.; Fairfield, Heather E.; Reinholdt, Laura G.; Brodsky, Gary L.; Davizon-Castillo, Pavel; Ginsburg, David; Di Paola, Jorge

2016-01-01

During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp) deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS), an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps) exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10−7). Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains. PMID:26950939

Spontaneous 8bp Deletion in Nbeal2 Recapitulates the Gray Platelet Syndrome in Mice.

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Kärt Tomberg

Full Text Available During the analysis of a whole genome ENU mutagenesis screen for thrombosis modifiers, a spontaneous 8 base pair (bp deletion causing a frameshift in exon 27 of the Nbeal2 gene was identified. Though initially considered as a plausible thrombosis modifier, this Nbeal2 mutation failed to suppress the synthetic lethal thrombosis on which the original ENU screen was based. Mutations in NBEAL2 cause Gray Platelet Syndrome (GPS, an autosomal recessive bleeding disorder characterized by macrothrombocytopenia and gray-appearing platelets due to lack of platelet alpha granules. Mice homozygous for the Nbeal2 8 bp deletion (Nbeal2gps/gps exhibit a phenotype similar to human GPS, with significantly reduced platelet counts compared to littermate controls (p = 1.63 x 10-7. Nbeal2gps/gps mice also have markedly reduced numbers of platelet alpha granules and an increased level of emperipolesis, consistent with previously characterized mice carrying targeted Nbeal2 null alleles. These findings confirm previous reports, provide an additional mouse model for GPS, and highlight the potentially confounding effect of background spontaneous mutation events in well-characterized mouse strains.

Thromboxane A(2 receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

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Tomohiro Yokota

Full Text Available Ductus arteriosus (DA closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

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

2017-06-01

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p platelet count and IPC (both p-values Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 platelet turnover and COX-1 expression (all p-values platelet turnover and COX-1 and COX-2 expressions (all p-values platelet turnover in ITP patients (all p-values 0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in

Blood platelet kinetics and platelet transfusion.

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Aster, Richard H

2013-11-01

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

Relationship Between Platelet and Urinary 8‐Iso‐PGF2α Levels in Subjects With Different Degrees of NOX2 Regulation

Science.gov (United States)

Carnevale, Roberto; Iuliano, Luigi; Nocella, Cristina; Bartimoccia, Simona; Trapè, Stefano; Russo, Roberta; Gentile, Maria Cristina; Cangemi, Roberto; Loffredo, Lorenzo; Pignatelli, Pasquale; Violi, Francesco

2013-01-01

Background Urinary 8‐iso‐PGF2α, a marker of oxidative stress, is influenced by the activation of NOX2. It is unclear if platelets 8‐iso‐PGF2α contribute to urinary 8‐iso‐PGF2α. Methods and Results In a cross‐sectional study, platelet, urinary, and serum 8‐iso‐PGF2α were determined in subjects with downregulation (X‐linked chronic granulomatous disease [X‐CGD], n=25) and upregulation (type II diabetic patients [T2D], n=121) of NOX2 and 153 controls matched for sex and age. In diabetic patients (n=18), the above variables were repeated before and after 7 days treatment with 100 mg/day aspirin or 100 mg/day aspirin plus 40 mg/day atorvastatin. In vitro study was performed to see the contribution of blood cells to serum 8‐iso‐PGF2α. Compared with controls, X‐CGD patients had lower platelet, serum, and urinary 8‐iso‐PGF2α values; conversely, diabetic patients had higher values of 8‐iso‐PGF2α compared with controls. Urinary 8‐iso‐PGF2α significantly correlated with both platelet and serum 8‐iso‐PGF2α in the 2 cohorts. A parallel increase of platelet, serum, and urinary 8‐iso‐PGF2α by aspirin and a parallel decrease by aspirin plus atorvastatin were detected in the interventional study. In vitro study demonstrated that platelets contribute to 37% of serum 8‐iso‐PGF2α and that only 13% of it is of extravascular origin. Conclusions The study suggests that NOX2 contributes to the formation of 8‐iso‐PGF2α in both platelets and urine. The direct correlation between platelet and urinary 8‐iso‐PGF2α suggests that, at least partly, urinary 8‐iso‐PGF2α reflects platelet 8‐iso‐PGF2α production. Analysis of serum 8‐iso‐PGF2α may represent a novel tool to investigate the production of 8‐iso‐PGF2α by blood cells including platelets. Clinical Trial Registration URL: ClinicalTrials.gov. Unique Identifier: NCT01250340. PMID:23770972

Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2.

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Satoshi Takagi

Full Text Available The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus-CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus-CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet-tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma.

Use of an anti-platelet monoclonal antibody F (ab')2 fragment for imaging thrombus

International Nuclear Information System (INIS)

Loutfi, I.; Stuttle, A.W.J.; Peters, A.M.; George, P.; Lavender, J.P.; Lumley, P.

1990-01-01

Ten patients with suspected thrombus have been studied using 111 In-labelled F (ab')2 fragments of P256, a monoclonal antibody which recognizes an epitope on the platelet membrane glycoprotein IIb/IIIa complex. The F (ab')2 fragment was radiolabelled with 111 In via diethylenetri-aminepentamacetic acid to give a specific activity of up to 190 MBq (5mCi) mg - 1 without impairment of immunoreactivity. In vitro platelet aggregation studies showed that the F (ab')2 fragment caused less platelet aggregation than the whole antibody on a molar ratio and was without significant effect upon the sensitivity of platelets to a range of aggregating agents. Platalets were labelled in ten patients by intravenous injection of approximately 100 μg P256 F (ab')2. Of the ten patients studies, six showed localization of activity consistent with platelet accumulation. Localization was clearly seen when associated with thrombus of the lower limbs (three patients: deep vein thrombosis; one patient: aortofemoral graft), and was apparent although less marked in two other cases, one of aortic aneurysm and one of carotid stenosis. Use of radiolabelled P256 F (ab')2 offers a means of non-invasive detection of thrombus which, from in vitro studies, would appear to have less direct effect of platelet behaviour than the whole antibody. (author). 9 refs. 8 figs. 1 tab

Assessment of canine autologous platelet-rich plasma produced with a commercial centrifugation and platelet recovery kit.

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Frye, Chris W; Enders, Andrew; Brooks, Marjory B; Struble, Angela M; Wakshlag, Joseph J

2016-01-01

To characterize the cellular composition (platelets, erythrocytes, and leukocytes) and confirm reproducibility of platelet enrichment, as well as determine the platelet activation status in the final product of a commercial platelet-rich plasma kit using canine blood. Venous blood from 20 sedated client-owned dogs was used to prepare platelet-rich plasma (PRP) from a commercial kit. Complete blood counts were performed to determine erythrocyte, leukocyte, and platelet numbers in both whole blood (WB) and resultant PRP. The WB and PRP samples from jugular (fast collection) and cephalic (slow collection) venipuncture were also compared. P-selectin externalization was measured in WB and PRP samples from 15 of 20 dogs. This commercial kit produced an average percent recovery in platelets of 64.7 ± 17.4; erythrocytes of 3.7 ± 0.8, and leukocytes of 31.6 ± 10.0. Neutrophil, monocyte, and lymphocyte percent recovery was 19.6 ± 7.2, 44.89 ± 19.8, and 57.5 ± 10.6, respectively. The recovery of platelets from jugular venipuncture (59.7 ± 13.6%) was lower than from cephalic recovery (68.8 ± 19.1%). The mean percent P-Selectin externalization for WB, PRP, and PRP with thrombin was 25.5 ± 30.9, 4.5 ± 6.4, and 90.6 ± 4.4 respectively. Cellular reproducibility of this kit was confirmed and platelets were concentrated within autologous serum. Additionally, measurements of P-selectin externalization showed that platelets are inactive in PRP unless stimulated to degranulate.

A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

OpenAIRE

Mustafa Tunalı; Hakan Özdemir; Zafer Küçükodacı; Serhan Akman; Emre Yaprak; Hülya Toker; Erhan Fıratlı

2014-01-01

We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn us...

Storage of platelets: effects associated with high platelet content in platelet storage containers.

Science.gov (United States)

Gulliksson, Hans; Sandgren, Per; Sjödin, Agneta; Hultenby, Kjell

2012-04-01

A major problem associated with platelet storage containers is that some platelet units show a dramatic fall in pH, especially above certain platelet contents. The aim of this study was a detailed investigation of the different in vitro effects occurring when the maximum storage capacity of a platelet container is exceeded as compared to normal storage. Buffy coats were combined in large-volume containers to create primary pools to be split into two equal aliquots for the preparation of platelets (450-520×10(9) platelets/unit) in SSP+ for 7-day storage in two containers (test and reference) with different platelet storage capacity (n=8). Exceeding the maximum storage capacity of the test platelet storage container resulted in immediate negative effects on platelet metabolism and energy supply, but also delayed effects on platelet function, activation and disintegration. Our study gives a very clear indication of the effects in different phases associated with exceeding the maximum storage capacity of platelet containers but throw little additional light on the mechanism initiating those negative effects. The problem appears to be complex and further studies in different media using different storage containers will be needed to understand the mechanisms involved.

Platelets of patients with chronic kidney disease demonstrate deficient platelet reactivity in vitro

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van Bladel Esther R

2012-09-01

Full Text Available Abstract Background In patients with chronic kidney disease studies focusing on platelet function and properties often are non-conclusive whereas only few studies use functional platelet tests. In this study we evaluated a recently developed functional flow cytometry based assay for the analysis of platelet function in chronic kidney disease. Methods Platelet reactivity was measured using flow cytometric analysis. Platelets in whole blood were triggered with different concentrations of agonists (TRAP, ADP, CRP. Platelet activation was quantified with staining for P-selectin, measuring the mean fluorescence intensity. Area under the curve and the concentration of half-maximal response were determined. Results We studied 23 patients with chronic kidney disease (9 patients with cardiorenal failure and 14 patients with end stage renal disease and 19 healthy controls. Expression of P-selectin on the platelet surface measured as mean fluorescence intensity was significantly less in chronic kidney disease patients compared to controls after maximal stimulation with TRAP (9.7 (7.9-10.8 vs. 11.4 (9.2-12.2, P = 0.032, ADP (1.6 (1.2-2.1 vs. 2.6 (1.9-3.5, P = 0.002 and CRP (9.2 (8.5-10.8 vs. 11.5 (9.5-12.9, P = 0.004. Also the area under the curve was significantly different. There was no significant difference in half-maximal response between both groups. Conclusion In this study we found that patients with chronic kidney disease show reduced platelet reactivity in response of ADP, TRAP and CRP compared to controls. These results contribute to our understanding of the aberrant platelet function observed in patients with chronic kidney disease and emphasize the significance of using functional whole blood platelet activation assays.

Platelet transfusions reduce fibrinolysis but do not restore platelet function during trauma hemorrhage.

Science.gov (United States)

Vulliamy, Paul; Gillespie, Scarlett; Gall, Lewis S; Green, Laura; Brohi, Karim; Davenport, Ross A

2017-09-01

Platelets play a critical role in hemostasis with aberrant function implicated in trauma-induced coagulopathy. However, the impact of massive transfusion protocols on platelet function during trauma hemorrhage is unknown. The aim of this study was to characterize the effects of platelet transfusion on platelet aggregation and fibrinolytic markers during hemostatic resuscitation. Trauma patients enrolled into the prospective Activation of Coagulation and Inflammation in Trauma study between January 2008 and November 2015 who received at least four units of packed red blood cells (PRBCs) were included. Blood was drawn in the emergency department within 2 hours of injury and at intervals after every four units of PRBCs transfused. Platelet aggregation was assessed in whole blood with multiple electrode aggregometry. Plasma proteins were quantified by enzyme-linked immunosorbent assay. Of 161 patients who received four or more PRBCs as part of their initial resuscitation, 44 received 8 to 11 units and 28 received 12 units or more. At each timepoint during bleeding, platelet aggregation was similar in patients who had received a platelet transfusion compared with those who had only received other blood products (p > 0.05 for all timepoints). Platelet transfusion during the four PRBC intervals was associated with a decrease in maximum lysis on rotational thromboelastometry (start of interval, 6% [2-12] vs. end of interval, 2% [0-5]; p = 0.001), an increase in plasminogen activator inhibitor-1 (start of interval, 35.9 ± 14.9 vs. end of interval, 66.7 ± 22.0; p = 0.007) and a decrease in tissue plasminogen activator (start of interval, 26.2 ± 10.5 vs. end of interval, 19.0 +/- 5.1; p = 0.04). No statistically significant changes in these parameters occurred in intervals which did not contain platelets. Current hemostatic resuscitation strategies do not appear to restore platelet aggregation during active hemorrhage. However, stored platelets may attenuate fibrinolysis

Superoxide dismutase of human platelets

International Nuclear Information System (INIS)

Kimura, Akiro; Fujimura, Kingo; Kuramoto, Atsushi

1979-01-01

Superoxide dismutase (S.O.D.) is the enzyme to protect from destructive effect of superoxide (O 2 -) produced in many metabolic pathways related to oxygen. The purpose of this study was to investigate the possibility that S.O.D. may play an important role in the platelet function. The cytoplasmic and mitochondrial S.O.D. has been investigated spectrophotometrically and gel electrophoretically in human platelets from eleven patients of chronic myelogenous leukemia (CML) and three patients of primary thrombocythemia (P.Th.). Neither deficiency nor abnormality of cytoplasmic and mitochondrial S.O.D. has been found electrophoretically in any case compared to normal platelets. However, the total activity from three of the CML patients and one of the P.Th. patients were above 3 unit/mg platelet protein (normal subject: 2.11 - 2.70 unit/mg protein), suggesting the possibility either that more O 2 -production occurs in the platelets or that rather little O 2 -production due to much O 2 -deprivation by the increased S.O.D. The S.O.D. activity of human platelets has been also investigated in several conditions, where much O 2 -generation might occur in platelets. Sodium fluoride (2 mM), which increases platelet O 2 -production about 3 fold, had no effect on platelet S.O.D. The aggregated platelets induced by ADP (10 -5 M), epinephrin (50 μg/ml), ristocetin (1.5 mg/ml) or collagen (1 - 20 μg/ml) had no increase of S.O.D. activity compared to that from non aggregated platelets. X-ray irradiation (1,000 - 20,000R) had not induced its activity increase or decrease. These findings indicated the induction of platelet S.O.D. was not brought about under these conditions. (author)

Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression

International Nuclear Information System (INIS)

Mori, Hideki; Koyama, Tsukasa; Yamashita, Itaru

1991-01-01

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets. This method involves the measurement of the accumulation of [ 3 H]-inositol-1-phosphate (IP-1) as an index of Pl hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression

Dose- and time-related platelet response with apheresis platelet concentrates and pooled platelets

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Mohammad Mizanur Rahman

2017-02-01

Full Text Available This study was carried out to compare the post-transfusion platelet increment between the apheresis platelet concentrate (n=74 and pooled platelets (n=54. Pre- and post-transfusion platelet count of the recipient were carried out by automated hematology analyzer. In apheresis platelet concentrate group, the mean 24 hours post-transfusion platelet increment was 47 x 109/L which was statistically significant (p<0.001. On the other hand, in pooled platelets group, the mean 24 hours post–transfusions platelet count increment was 11.0 x 109/L which was also statistically significant (p<0.001. This study concluded that the transfusion of apheresis platelet concentrate was more useful than the transfusion of pooled platelets in terms of platelet count increment and requirement of donor.

Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

Science.gov (United States)

Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

2011-01-01

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

Analysis of vesicle fluid following the sting of the lionfish Pterois volitans.

Science.gov (United States)

Auerbach, P S; McKinney, H E; Rees, R S; Heggers, J P

1987-01-01

Fluid aspirated from blisters following a lionfish (Pterois volitans) sting was analyzed utilizing combined capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Analysis for prostaglandin F2 alpha demonstrated 16.91 ng/ml, for prostaglandin E2 0.143 ng/ml, for 6-keto-prostaglandin F1 alpha less than 0.1 ng/ml (nondetectable) and for thromboxane B2 1.65 ng/ml. Platelet aggregation studies showed that blister fluid caused aggregation of isolated platelets only, which was inhibited by heat treatment or by the presence of normal donor plasma.

Measurement of platelet aggregation, independently of patient platelet count

DEFF Research Database (Denmark)

Vinholt, P J; Frederiksen, H; Hvas, A-M

2017-01-01

with collagen-related peptide). Platelet aggregation had a negative predictive value of 100% for a bleeding tendency among patients. Conclusion The established platelet aggregation assay was applicable for thrombocytopenic patients, and improved the identification of bleeding risk.......Essentials •Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. •A flow cytometric platelet aggregation test independent of the patient platelet count was made. •Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer....... •High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients. Summary Background Methods for testing platelet aggregation in thrombocytopenia are lacking. Objective To establish a flow-cytometric test of in vitro platelet aggregation independently of the patient's platelet count...

A novel platelet concentrate: titanium-prepared platelet-rich fibrin.

Science.gov (United States)

Tunalı, Mustafa; Özdemir, Hakan; Küçükodacı, Zafer; Akman, Serhan; Yaprak, Emre; Toker, Hülya; Fıratlı, Erhan

2014-01-01

We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF). The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun's leukocyte- and platelet-rich fibrin (L-PRF) method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF) was processed with a scanning electron microscope (SEM). The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

A Novel Platelet Concentrate: Titanium-Prepared Platelet-Rich Fibrin

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Mustafa Tunalı

2014-01-01

Full Text Available We developed a new product called titanium-prepared platelet-rich fibrin (T-PRF. The T-PRF method is based on the hypothesis that titanium may be more effective in activating platelets than the silica activators used with glass tubes in Chouckroun’s leukocyte- and platelet-rich fibrin (L-PRF method. In this study, we aimed to define the structural characteristics of T-PRF and compare it with L-PRF. Blood samples were collected from 10 healthy male volunteers. The blood samples were drawn using a syringe. Nine milliliters was transferred to a dry glass tube, and 9 mL was transferred to a titanium tube. Half of each clot (i.e., the blood that was clotted using T-PRF or L-PRF was processed with a scanning electron microscope (SEM. The other half of each clot was processed for fluorescence microscopy analysis and light microscopy analysis. The T-PRF samples seemed to have a highly organized network with continuous integrity compared to the other L-PRF samples. Histomorphometric analysis showed that T-PRF fibrin network covers larger area than L-PRF fibrin network; also fibrin seemed thicker in the T-PRF samples. This is the first human study to define T-PRF as an autogenous leukocyte- and platelet-rich fibrin product. The platelet activation by titanium seems to offer some high characteristics to T-PRF.

Platelet collection efficiencies of three different platelet-rich plasma preparation systems.

Science.gov (United States)

Aydin, Fatma; Pancar Yuksel, Esra; Albayrak, Davut

2015-06-01

Different systems have been used for the preparation of platelet-rich plasma (PRP), but platelet collection efficiencies of these systems are not clear. To evaluate the platelet collection efficiencies of three different PRP preparation systems. Blood samples were obtained from the same 16 volunteers for each system. The samples were centrifuged and PRP was prepared by three systems. The ratio of the total number of platelets in PRP to the total number of platelets of the venous blood sample of the patient expressed in percentage was named as platelet collection efficiency and calculated for each system. Mean platelet collection efficiencies were 66.6 (min: 56.9, max: 76.9), 58.3 (min: 27.3, max: 102.8), 50.8 (min: 27.2, max: 73) for top and bottom bag system, system using citrated tube, and the system using tube with Ficoll and cell extraction kit, respectively. Statistically significant difference was found only between the platelet collection efficiencies of systems using the tube with ficoll and cell extraction kit and the top and bottom bag system (p = 0.002). All three systems could be used for PRP preparation, but top and bottom bag system offers a slight advantage over the system using Ficoll and cell extraction kit regarding the platelet collection efficiency.

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

International Nuclear Information System (INIS)

Kao, K.J.

1988-01-01

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I-125-labeled platelets showed that only beta 2-M but not other I-125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I-125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins

Effect of pretreatment with omega-3 polyunsaturated fatty acids (PUFAs on hematological parameters and platelets aggregation in patients during elective coronary artery bypass grafting

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Veljović Milić

2013-01-01

Full Text Available Bacground/Aim. Using omega-3 polyunsaturated fatty acids (PUFAs in coronary artery bypass graft surgery (CABG could provide protection against ischemicreperfusion damage, prevention of postoperative arrhythmia and attenuation of inflammatory response. However, omega-3 PUFAs inhibit cyclooxygenase (and thus decrease the synthesis of thromboxane A2 from arachidonic acid in platelets, which leads to decreased platelet aggregation. In cardiac surgery it is necessary to achieve a balance between inhibition and full platelets function. It is as well as important to closely follow hematological parameters, impaired by CABG itself. Therefore, the aim of the study was to establish the effects of pretreatment with omega- PUFAs on hematological parameters and plateletes aggregation in patients with elective CABG. Methods. This prospective, randomized, placebo-controlled, single-center trial was performed on parallel groups. The patients (n = 40 undergoing elective CABG were randomized receiving preoperative intravenous omega-3 PUFAs (Omegaven® 10% infusion (the PUFAs group or the same volume of 0.9% saline solution infusion (the control group. Infusion was given a day before surgery and repeated four hours before starting extracorporeal circulation (CPB via the peripheral vein at single doses of 100 mL (25 mL/h. Platelet function analysis was performed using multiple electrode aggregometry (MEA, multiplate-analyzer before starting CPB and 2 h postoperatively for the patients of both groups. Results. There were no clinically relevant differences in baseline characteristics between the groups. Hematological parameters were not significantly different between the groups pre-, intra- and postoperatively. During the first 24 h after surgery, the loss of blood was similar in the PUFAs and the control group (680 ± 274 mL and 608 ± 210 mL, respectively; p = 0.356. Postoperatively, platelet aggregation was not significantly different between the PUFAs and the

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

Science.gov (United States)

Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

2017-10-11

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

Platelet count and platelet indices in women with preeclampsia.

Science.gov (United States)

AlSheeha, Muneera A; Alaboudi, Rafi S; Alghasham, Mohammad A; Iqbal, Javed; Adam, Ishag

2016-01-01

Although the exact pathophysiology of preeclampsia is not completely understood, the utility of different platelets indices can be utilized to predict preeclampsia. To compare platelet indices, namely platelet count (PC), mean platelet volume (MPV), platelet distribution width (PDW), and PC to MPV ratio in women with preeclampsia compared with healthy controls. Qassim Hospital, Kingdom of Saudi Arabia. A case-control study. Sixty preeclamptic women were the cases and an equal number of healthy pregnant women were the controls. There was no significant difference in age, parity, and body mass index between the study groups. Sixteen and 44 of the cases were severe and mild preeclampsia, respectively. There was no significant difference in PDW and MPV between the preeclamptic and control women. Both PC and PC to MPV ratios were significantly lower in the women with preeclampsia compared with the controls. There was no significant difference in the PC, PDW, MPV, and PC to MPV ratio when women with mild and severe preeclampsia were compared. Using receiver operating characteristic (ROC) curves, the PC cutoff was 248.0×10 3 /µL for diagnosis of pre-eclampsia ( P =0.019; the area under the ROC curve was 62.4%). Binary regression suggests that women with PC preeclampsia (odds ratio =2.2, 95% confidence interval =1.08-4.6, P =0.03). The PC/MPV cutoff was 31.2 for diagnosis of preeclampsia ( P =0.035, the area under the ROC curve was 62.2%). PC preeclampsia.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

Science.gov (United States)

Cailleteau, C; Liagre, B; Battu, S; Jayat-Vignoles, C; Beneytout, J L

2008-09-01

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

The content of bone morphogenetic proteins in platelets varies greatly between different platelet donors

International Nuclear Information System (INIS)

Kalen, Anders; Wahlstroem, Ola; Linder, Cecilia Halling; Magnusson, Per

2008-01-01

Platelet derivates and platelet rich plasma have been used to stimulate bone formation and wound healing because of the rich content of potent growth factors. However, not all reports have been conclusive since some have not been able to demonstrate a positive effect. We investigated the interindividual variation of bone morphogenetic proteins (BMPs) in platelets from healthy donors, and the pH-dependent effect on the release of BMPs in preparations of lysed platelets in buffer (LPB). Platelet concentrates from 31 healthy donors were prepared in pH 4.3 and pH 7.4 buffers and investigated with respect to BMP-2, -4, -6, and -7. BMP-2 and BMP-4 were significantly more common in acidic LPBs in comparison with neutral preparations. We also observed a considerable variation among platelet donors with respect to the release of BMPs at pH 4.3 and 7.4. In conclusion, a considerable variation was found among platelet donors, which may be of importance considering the ambiguous results previously reported on osteoblast proliferation and differentiation

Radiolabeled platelets

International Nuclear Information System (INIS)

Datz, F.L.; Taylor, A.T.

1986-01-01

Initial interest in developing techniques to radiolabel platelets was spurred by the lack of an accurate method for measuring platelet life span in both normals and in thrombocytopenic patients. Early investigators could obtain only rough estimates of platelet life spans by monitoring the platelet counts of thrombocytopenic patients undergoing platelet transfusions. Labels were also sought that would allow imaging of platelets in vivo in order to better understand the pathophysiology of atherosclerosis, thrombophlebitis, and clotting disorders, and to improve the clinical diagnosis of these diseases. Two types of platelet labels were investigated: cohort (pulse) labels and random labels. Cohort labels are taken up by megakaryocytes in the bone marrow and incorporated in the DNA and other components of the forming platelet. In theory, only freshly released platelets of a uniform age are labeled. Random labels, on the other hand, tag platelets in the peripheral blood, labeling platelets of all ages

Blood platelet kinetics and platelet transfusion

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Aster, Richard H.

2013-01-01

The discovery of citrate anticoagulant in the 1920s and the development of plastic packs for blood collection in the 1960s laid the groundwork for platelet transfusion therapy on a scale not previously possible. A major limitation, however, was the finding that platelet concentrates prepared from blood anticoagulated with citrate were unsuitable for transfusion because of platelet clumping. We found that this could be prevented by simply reducing the pH of platelet-rich plasma to about 6.5 pr...

Platelet Arachidonic Acid Deficiency May Contribute to Abnormal Platelet Function During Parenteral Fish Oil Monotherapy in a Piglet Model.

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Turner, Justine M; Field, Catherine J; Goruk, Sue; Wizzard, Pamela; Dicken, Bryan J; Bruce, Aisha; Wales, Paul W

2016-05-01

Fish oil monotherapy has been an advance for treating intestinal failure-associated liver disease (IFALD). However, such patients are at risk of bleeding complications from liver disease and because fish oil can inhibit thrombosis. We have previously reported abnormal platelet function in neonatal piglets given fish oil monotherapy during parenteral nutrition (PN). The purpose of this study was to determine if abnormal fatty acid composition of the platelets could explain the prior observed antiplatelet effect. Neonatal piglets were assigned to 2 treatments: PN with fish oil monotherapy (FO; n = 4) or PN with soy oil (SO; n = 5). On day 14, plasma was collected and platelets isolated by centrifuging. The fatty acid content in plasma and platelet plug were measured using gas liquid chromatography and compared with controls (CON; n = 5). The arachidonic acid (AA) content in the FO group was on average half that of the SO group, in both the platelets (FO, 3.5% vs SO, 7.6%; P = .021; CON, 4.5%-11%) and the plasma (FO, 3.8% vs SO, 9.2%; P = .002; CON, 6.1%-9.5%). No bleeding complications were observed for any piglets during PN treatment. Using platelet mapping, we have previously shown that neonatal piglets given fish oil monotherapy have abnormal platelet function in the AA pathway. This report demonstrates that such an abnormality can be explained by platelet AA deficiency. Platelet mapping and platelet fatty acid analysis should be undertaken in human infants treated with fish oil monotherapy during PN. © 2015 American Society for Parenteral and Enteral Nutrition.

Measurement of urinary 11-dehydro-thromboxane B2 excretion in dogs with gastric dilatation-volvulus.

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Baltzer, Wendy I; McMichael, Maureen A; Ruaux, Craig G; Noaker, Laura; Steiner, Jörg M; Williams, David A

2006-01-01

To measure 11-dehydro-thromboxane B2 (11-dTXB2) in urine of healthy control dogs, dogs undergoing ovariohysterectomy, and dogs with gastric dilatation-volvulus (GDV) and assess the relationship between urinary 11-dTXB2 concentrations in dogs with GDV and postoperative outcomes. Urine samples from 15 nonsurgical control dogs, 12 surgical control dogs, and 32 dogs with GVD. Urine samples were obtained from healthy pet dogs (ie, nonsurgical control dogs), dogs undergoing ovariohysterectomy at anesthetic induction and 1 hour following surgery (ie, surgical control dogs), and dogs with GDV at hospital admission and 1 hour following surgical derotation of the stomach (ie, GDV dogs). Urinary 11-dTXB2 concentrations were determined with an ELISA and normalized to urinary creatinine (Cr) concentrations by calculation of the 11-dTXB2 -to-Cr ratio. Differences in median 11-dTXB2 -to-Cr ratios among dogs and before and after surgery were analyzed. Urinary 11-dTXB2-to-Cr ratios did not differ between nonsurgical control dogs and surgical control dogs before or after surgery. Urinary 11-dTXB2-to-Cr ratios were significantly higher in GDV dogs at the time of hospital admission and 1 hour after surgery, compared with those of nonsurgical control dogs. Postoperative urine samples from GDV dogs had significantly higher 11-dTXB2-to-Cr ratios than postoperative urine samples from surgical control dogs. Median urinary 11-dTXB2-to-Cr ratios increased significantly in GDV dogs that developed postoperative complications. Urinary 11-dTXB2 concentration is increased in GDV dogs at the time of hospital admission and after surgical derotation of the stomach, compared with that of healthy dogs. An increased urinary 11-dTXB2-to-Cr ratio following surgery is associated with an increased incidence of postoperative complications in dogs with GDV.

Increased mean platelet volume in type 2 diabetes mellitus

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Ezgi Coşkun Yenigün

2014-03-01

Full Text Available Objective: Platelet functions have important roles in the development of vascular complications in diabetic patients. Platelets with increased volume have increased activity compared to smaller ones; therefore, mean platelet volume (MPV is used as a marker for platelet activity. In the present study, we evaluated MPV in patients with type II diabetes mellitus (DM and its associations with diabetic microvascular and macrovascular complications. Methods: Consecutive type II diabetic patients were screened from outpatient clinic of Internal Medicine Department of Diskapı Yıldırım Beyazıt Education and Researsch Hospital, Ankara, Turkey. A total of 48 patients with type II DM and 30 age and gender matched healthy subjects constituted the study population. For all subjects a complete blood count including MPV, fasting blood glucose level and lipid parameters were studied. In diabetic patients, duration of diabetes and HbA1C level, presence of microvascular and macrovascular complications were noted additively. Mean platelet volume was compared between diabetic patients and healthy counterparents. Then, among diabetic patients, MPV was compared between the ones with and without microvascular and macrovascular complications. Results: Mean platelet volume was found significantly higher in diabetic patients compared to non-diabetic healthy subjects. Diabetic patients with at least one of the microvascular complications had significantly higher MPV than those without microvascular damage.Higher MPV levels have also been shown in diabetics with macrovascular complications compared to the ones without macrovascular disease. Conclusion: Mean platelet volume was found to be higher in type II diabetics and those having any of microvascular or macrovascular diabetic complications.

Blood clotting and platelets: a delicate balance

International Nuclear Information System (INIS)

Heyns, A. du P.; Loetter, M.G.; Badenhorst, P.N.

1988-01-01

The Medical Research Council Blood Platelet Research Unit has studied many of the facets of platelets. The research highlights of the Unit include the following: the identification of ADPase, an enzyme found in the blood vessel wall, which breaks down adenosine diphospate (ADP) to adenosine, and which inhibits platelet aggregation; the determination of the relationship between the biochemical activity of the vessel wall with the development of atherosclerosis; the development of a method to label platelets with the compound In-111-oxine; the use of labelled platelets to study platelets in the spleen: an investigation of the life-span of platelets, which revealed how aged platelets are removed from circulation, and recently a study of methods to demonstrate in vivo activation of platelets. 1 fig

Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPβ isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

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O'Sullivan, Aine G; Mulvaney, Eamon P; Kinsella, B Therese

2017-04-01

The prostanoid thromboxane (TX) A 2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA 2 /TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA 2 -TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA 2 /TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA 2 /TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA 2 /TP signalling axis in PCa, including potentially in CRPC. Copyright © 2017 Elsevier B.V. All rights reserved.

A Comparison of Platelet Count and Enrichment Percentages in the Platelet Rich Plasma (PRP) Obtained Following Preparation by Three Different Methods.

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Sabarish, Ram; Lavu, Vamsi; Rao, Suresh Ranga

2015-02-01

Platelet rich plasma (PRP) represents an easily accessible and rich source of autologous growth factors. Different manual methods for the preparation of PRP have been suggested. Lacuna in knowledge exists about the efficacy of PRP preparation by these different manual methods. This study was performed to determine the effects of centrifugation rate revolutions per minute (RPM) and time on the platelet count and enrichment percentages in the concentrates obtained following the three different manual methods of PRP preparation. In vitro experimental study. This was an experimental study in which platelet concentration was assessed in the PRP prepared by three different protocols as suggested by Marx R (method 1), Okuda K (method 2) and Landesberg R (method 3). A total of 60 peripheral blood samples, (n=20 per method) were obtained from healthy volunteers. Baseline platelet count was assessed for all the subjects following which PRP was prepared. The platelet count in the PRP was determined using coulter counter (Sysmex XT 2000i). The mean of the platelet count obtained and their enrichment percentage were calculated and intergroup comparison was done (Tukey's HSD test). The number of platelets and enrichment percentage in PRP prepared by method 1 was higher compared to method 2 and method 3; this difference in platelet concentrates was found to be statistically significant (p < 0.05). The centrifugation rate and time appear to be important parameters, which influence the platelet yield. Method 1 which had lower centrifugation rate and time yielded a greater platelet count and enrichment percentage.

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

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Selheim, F; Holmsen, H; Vassbotn, F S

1999-08-15

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.

A radiolabeled antiglobulin test for crossmatching platelet transfusions

International Nuclear Information System (INIS)

Kickler, T.S.; Braine, H.G.; Ness, P.M.; Koester, A.; Bias, W.

1983-01-01

Despite the use of HLA-matched platelets for alloimmunized recipients, transfusion failures occur. In order to reduce these failures, researchers investigated the use of a radiolabeled antiglobulin technique for platelet crossmatching. The principle of the test is that of an indirect Coombs test using 125 I labeled goat anti-human IgG. Incompatibility is determined by calculating a radioactivity antiglobulin test (RAGT) index. Using this technique, researchers performed 89 crossmatches on 19 leukemic or aplastic patients who were refractory to random donor platelets and receiving varying degrees of HLA-matched platelets. Effectiveness of the transfusion was assessed from the posttransfusion corrected platelet count increment (CCI) determined at 1 and 20 hr. When the RAGT index was 1.9 or less, the mean CCI at 1 lhr was 17,570 +/- 7003/cu mm, n . 55. When the RAGT index was 2.0 or greater, the mean CCI was 4237 +/- 4100/cu mm, n . 34. At 20 hr when the RAGT index was 1.9 or less, the mean CCI was 8722 +/- 3143/cu mm, n . 33, and when the index was 2.0 or greater, the mean CCI was 571 +/- 1286/cu mm, n . 23. Using this technique, one false negative resulted. Nine positive crossmatches with good increments at 1 hr were found; at 20 hr, however, the survival of these units was zero. These data suggest that this method is a useful adjunct in the selection of platelets in the refractory patient

Enhanced Store-Operated Calcium Entry in Platelets is Associated with Peripheral Artery Disease in Type 2 Diabetes

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Weijie Xia

2015-11-01

Full Text Available Background/Aims: Platelet dysfunction plays an important role in thrombosis in diabetes with peripheral artery disease (PAD. Store-operated calcium entry (SOCE and stromal interaction molecule 1 (STIM1 regulate platelet activity by modulating calcium influx. We hypothesized that enhanced SOCE in platelets is associated with diabetes with PAD. Methods: We studied the activity of platelets from healthy participants and from type 2 diabetic patients. Platelet calcium influx and protein expression of STIM1 and sarcoendoplasmic reticulum Ca2+-ATPase 3 (SERCA3 were investigated. Results: Compared with platelets from diabetic patients without PAD, platelets from diabetic patients with PAD exhibited significantly increased SOCE . Menthol administration completely inhibited calcium influx in platelets from diabetic patients without PAD, but this effect was blunted in those from diabetic patients with PAD. Furthermore, the increase in SOCE was correlated with the ankle brachial index (ABI in diabetic patients. High glucose significantly up-regulated STIM1 and SERCA3 protein expression and induced the phosphorylation of phospholipase C (PLC in platelets from healthy participants. This effect was attenuated in the presence of menthol or U73122, an inhibitor of PLC. Similarly, significant increases in STIM1 and SERCA3 protein expression were found in platelets from diabetic patients compared to those from healthy participants. Conclusion: Platelets from diabetic patients with PAD exhibited enhanced Store-operated calcium influx, which was associated with elevated STIM1/SERCA3 expression via a PLC-dependent pathway and was inhibited by menthol.

Thromboxane A{sub 2} receptor signaling promotes liver tissue repair after toxic injury through the enhancement of macrophage recruitment

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Minamino, Tsutomu [Departments of Pharmacology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Departments of Gastroenterology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Ito, Yoshiya [Departments of Surgery, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Ohkubo, Hirotoki [Departments of Pharmacology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Departments of Surgery, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Hosono, Kanako; Suzuki, Tatsunori [Departments of Pharmacology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Sato, Takehito [Departments of Pharmacology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Departments of Gastroenterology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Ae, Takako; Shibuya, Akitaka [Departments of Gastroenterology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Sakagami, Hiroyuki [Departments of Anatomy, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Narumiya, Shuh [Department of Pharmacology, Kyoto University School of Medicine, Kyoto, 606-8315 (Japan); Koizumi, Wasaburo [Departments of Gastroenterology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan); Majima, Masataka, E-mail: mmajima@med.kitasato-u.ac.jp [Departments of Pharmacology, Kitasato University School of Medicine, Kanagawa 252-0374 (Japan)

2012-02-15

It is thought that thromboxane A{sub 2} (TxA{sub 2}) contributes to the progression of inflammation during acute hepatic injury; however, it is still unknown whether TxA{sub 2} is involved in liver repair. The objective of the present study was to examine the role of TxA{sub 2} receptor (TP) signaling in liver injury and repair in response to toxic injury. Carbon tetrachloride (CCl{sub 4}) was used to induce liver injury in TP knockout (TP{sup −/−}) mice and wild-type (WT) mice. In WT mice, serum levels of alanine aminotransferase (ALT) and the size of the necrotic area peaked at 24 and 48 h, respectively, and then declined. In TP{sup −/−} mice, the changes in ALT levels were similar to WT mice, but liver regeneration was impaired as evidenced by remained elevated levels of hepatic necrosis and by delayed hepatocyte proliferation, which was associated with the reduced expression of growth factors including interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and hepatocyte growth factor (HGF). In TP{sup −/−} mice, the accumulation of hepatic CD11b{sup +}/F4/80{sup +} macrophages in injured livers was attenuated, and the hepatic expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and its receptor, the C―C chemokine receptor (CCR2), was reduced compared to WT. Additionally, the application of the TP receptor agonist, U-46619, enhanced the expression of MCP-1/CCL2 and CCR2 in peritoneal macrophages, which was associated with increased levels of IL-6, TNFα and HGF. These results suggested that TP receptor signaling facilitates liver recovery following CCl{sub 4}-induced hepatotoxicity by affecting the expression of hepatotrophic growth factors, and through the recruitment of macrophages mediated by MCP-1/CCL2-CCR2 expression. -- Highlights: ► TP enhances liver regeneration by CCl{sub 4}. ► TP accumulates macrophages. ► TP up-regulates MCP-1.

YY-39, a tick anti-thrombosis peptide containing RGD domain.

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Tang, Jing; Fang, Yaqun; Han, Yajun; Bai, Xuewei; Yan, Xiuwen; Zhang, Yun; Lai, Ren; Zhang, Zhiye

2015-06-01

Ticks are obligatory blood feeding ectoparasites, which continuously attach to their hosts for 1-2 weeks. There are many biologically active compounds in tick salivary glands interfering host haemostatic system and to successfully obtain blood meal. Several platelet aggregation inhibitors have been identified from ticks. A family of conserved peptides, which were identified from transcriptome analysis of many tick salivary glands, were found to contain unique primary structure including predicted mature peptides of 39-47 amino acid residues in length and a Pro/Glu(P/E)-Pro/His(P/H)-Lys-Gly-Asp(RGD) domain. Given their unique structure and RGD domain, they are considered a novel family of disintegrins that inhibit platelet aggregation. One of them (YY-39) was tested for its effects on platelets and thrombosis in vivo. YY-39 was found effectively to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and thromboxane A2 (TXA2). Furthermore, YY-39 blocked platelet adhesion to soluble collagen and bound to purified GPIIb/IIIa in a dose-dependent manner. In in vivo experiments, YY-39 reduced thrombus weight effectively in a rat arteriovenous shunt model and inhibited thrombosis in a carrageenan-induced mouse tail thrombosis model. Combined with their prevalence in ticks and platelet inhibitory functions, this family of peptides might be conserved tick anti-haemostatic molecules. Copyright © 2014 Elsevier Inc. All rights reserved.

Reduced Antiplatelet Effect of Aspirin Does Not Predict Cardiovascular Events in Patients With Stable Coronary Artery Disease.

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

2017-08-05

Increased platelet aggregation during antiplatelet therapy may predict cardiovascular events in patients with coronary artery disease. The majority of these patients receive aspirin monotherapy. We aimed to investigate whether high platelet-aggregation levels predict cardiovascular events in stable coronary artery disease patients treated with aspirin. We included 900 stable coronary artery disease patients with either previous myocardial infarction, type 2 diabetes mellitus, or both. All patients received single antithrombotic therapy with 75 mg aspirin daily. Platelet aggregation was evaluated 1 hour after aspirin intake using the VerifyNow Aspirin Assay (Accriva Diagnostics) and Multiplate Analyzer (Roche; agonists: arachidonic acid and collagen). Adherence to aspirin was confirmed by serum thromboxane B 2 . The primary end point was the composite of nonfatal myocardial infarction, ischemic stroke, and cardiovascular death. At 3-year follow-up, 78 primary end points were registered. The primary end point did not occur more frequently in patients with high platelet-aggregation levels (first versus fourth quartile) assessed by VerifyNow (hazard ratio: 0.5 [95% CI, 0.3-1.1], P =0.08) or Multiplate using arachidonic acid (hazard ratio: 1.0 [95% CI, 0.5-2.1], P =0.92) or collagen (hazard ratio: 1.4 [95% CI, 0.7-2.8], P =0.38). Similar results were found for the composite secondary end point (nonfatal myocardial infarction, ischemic stroke, stent thrombosis, and all-cause death) and the single end points. Thromboxane B 2 levels did not predict any end points. Renal insufficiency was the only clinical risk factor predicting the primary and secondary end points. This study is the largest to investigate platelet aggregation in stable coronary artery disease patients receiving aspirin as single antithrombotic therapy. We found that high platelet-aggregation levels did not predict cardiovascular events. © 2017 The Authors. Published on behalf of the American Heart

Platelet destruction in autoimmune thrombocytopenic purpura: kinetics and clearance of indium-111-labeled autologous platelets

International Nuclear Information System (INIS)

Stratton, J.R.; Ballem, P.J.; Gernsheimer, T.; Cerqueira, M.; Slichter, S.J.

1989-01-01

Using autologous 111 In-labeled platelets, platelet kinetics and the sites of platelet destruction were assessed in 16 normal subjects (13 with and three without spleens), in 17 studies of patients with primary autoimmune thrombocytopenic purpura (AITP), in six studies of patients with secondary AITP, in ten studies of patients with AITP following splenectomy, and in five thrombocytopenic patients with myelodysplastic syndromes. In normal subjects, the spleen accounted for 24 +/- 4% of platelet destruction and the liver for 15 +/- 2%. Untreated patients with primary AITP had increased splenic destruction (40 +/- 14%, p less than 0.001) but not hepatic destruction (13 +/- 5%). Compared with untreated patients, prednisone treated patients did not have significantly different spleen and liver platelet sequestration. Patients with secondary AITP had similar platelet counts, platelet survivals, and increases in splenic destruction of platelets as did patients with primary AITP. In contrast, patients with myelodysplastic syndromes had a normal pattern of platelet destruction. In AITP patients following splenectomy, the five nonresponders all had a marked increase (greater than 45%) in liver destruction compared to five responders (all less than 40%). Among all patients with primary or secondary AITP, there was an inverse relationship between the percent of platelets destroyed in the liver plus spleen and both the platelet count (r = 0.75, p less than 0.001) and the platelet survival (r = 0.86, p less than 0.001). In a stepwise multiple linear regression analysis, total liver plus spleen platelet destruction, the platelet survival and the platelet turnover were all significant independent predictors of the platelet count. Thus platelet destruction is shifted to the spleen in primary and secondary AITP. Failure of splenectomy is associated with a marked elevation in liver destruction

A scalable, micropore, platelet rich plasma separation device.

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Dickson, Mary Nora; Amar, Levy; Hill, Michael; Schwartz, Joseph; Leonard, Edward F

2012-12-01

We have designed a novel, low energy platelet-rich-plasma (PRP) separator capable of producing 50 mL of PRP in 30 min, intended for military and emergency applications. Blood flows over a 3 mm length of sieve at high rates of shear. A plasma-platelet filtrate passes through the sieve's pores while erythrocytes remain. The filtrate is flowed over a second 3 mm length of smaller-pored sieve that withdraws plasma. Bulk blood volume is maintained by returning platelet-free plasma to the erythrocyte pool, enabling a nearly complete multi-pass platelet extraction. The total percentage of platelets extracted is:θ(T)=1-exp (-V(f)(T)Φ(P)/V) where V is the original plasma volume, V ( f )(T) is the total filtered volume, and ϕ ( P ) is platelet passage ratio (filtrate concentration/bulk average concentration) taken to be constant. Maximum θ(T) occurs at maximum V ( f )(T)× ϕ ( P ) Test microsieves, 3 mm long × 3 mm wide, were used. ϕ ( P ) values measured at various filtrate flow rates (20-100 uL/min) and utilizing various filter pore sizes (1.2-3.5 μm), was as high as 150 %. Maximum V ( f )(T)× ϕ ( P ) was achieved utilizing the 3.5 um filters at the highest flow rate, 100 uL/min. Erythrocyte leakages were always below 2,000/uL, far below the allowable limit stipulated by the American Association of Blood Banking. These data imply that a 13.7 cm(2) filter area is sufficient to achieve the target separation of 50 mL of platelet concentrate in 30 min. The filtration cartridge would consist of multiple microporous strips of 3 mm width arranged in parallel so that each element would see the conditions used in the prototype experiments presented here. Other microfiltration schemes suggest no method of scaling to practical levels.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

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Kanamarlapudi, Venkateswarlu; Owens, Sian E; Saha, Keya; Pope, Robert J; Mundell, Stuart J

2012-01-01

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

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Venkateswarlu Kanamarlapudi

Full Text Available Adenosine diphosphate (ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1 and P2Y(12 purinoceptors. Recently, we demonstrated that P2Y(1 and P2Y(12 purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6 in the internalization and function of P2Y(1 and P2Y(12 purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1 or P2Y(12 purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

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

International Nuclear Information System (INIS)

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

1990-01-01

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

Relationship between platelet phospholipid FA and mean platelet volume in healthy men.

Science.gov (United States)

Li, Duo; Turner, Alan; Sinclair, Andrew J

2002-09-01

Increased mean platelet volume (MPV) has been suggested as an independent risk factor for acute myocardial infarction and the increased reactivity of large platelets. The aim of this study was to investigate the correlation between platelet phospholipid (PL) PUFA composition and MPV in 139 free-living healthy men ages 20-55 yr (vegans, n = 18; ovolacto vegetarians, n = 43; moderate meat-eaters, n = 60; and high meateaters, n = 18). Each subject completed a semiquantitative Food Frequency Questionnaire and gave a blood sample. Platelet PL FA composition and MPV were determined by standard methods. MPV was significantly greater in the vegans than in the ovolacto vegetarian, moderate, or high meat-eater groups (P vegan and ovolacto vegetarian groups had significantly higher platelet PL 18:2n-6 and 22:4n-6, and lower 20:5n-3 and 22:6n-3 compared with the moderate and high meat-eater groups. The vegans demonstrated a significant reduction in 20:4n-6 and 22:5n-3 compared with the ovolacto vegetarian, high meat-eater, and moderate meat-eater groups. Bivariate analysis results showed that MPV was significantly positively correlated with platelet PL 18:2n-6 (P = 0.048) and negatively correlated with 20:3n-6 (P = 0.02), 20:5n-3 (P = 0.005), and 22:5n-3 (P< 0.0001), respectively. In a multiple linear regression analysis, after controlling for potential confounding factors such as dietary group, age, exercise, body mass index, and dietary polyunsaturated and saturated fat, cholesterol, carbohydrate, and fiber intake, the MPV was still strongly negatively correlated with platelet PL 20:3n-6 (P = 0.003) and 22:5n-3 (P = 0.001). The present data suggest that 22:5n-3 and 20:3n-6 may play a role in the structural function of the platelet membrane.

Indium-111 platelet imaging for detection of platelet deposition in abdominal aneurysms and prosthetic arterial grafts

International Nuclear Information System (INIS)

Ritchie, J.L.; Stratton, J.R.; Thiele, B.; Haminton, G.W.; Warrick, L.N.; Huang, T.W.; Harker, L.A.

1981-01-01

Thirty-four platelet imaging studies were performed in 23 patients to determine whether platelet deposition could be detected in patients with vascular aneurysms (18 patients) or in patients in whom Dacron prosthetic grafts had been placed (5 patients). In patients in whom abnormal platelet deposition was detected, the effect of administration of platelet-active drugs on platelet deposition was examined. Of the 18 patients with an aneurysm, 12 had equivocally positive studies on initial imaging and 2 had equivocally positive images. Of five patients with Dacron arterial grafts in place, four had diffuse platelet deposition in the grafts; the fifth patient had a platelet deposition only in a pseudoaneurysm. Eight patients with an abdominal aneurysm and positive or equivocally positive baseline images were restudied during platelet-active drug therapy either with aspirin plus dipyridamole (seven patients) or with sulfinpyrazone (four patients). No patient studied during treatment with aspirin plus dipyridamole had detectably decreased platelet deposition compared with baseline determinations. In contrast, two of four patients studied while receiving sulfinpyrazone showed decreased platelet deposition. Thus, platelet imaging may be of value for studying platelet physiology in vivo and for assessing platelet-active drugs and the thrombogenicity of prosthetic graft materials in human beings

Stimulus-response coupling in platelets

International Nuclear Information System (INIS)

Huang, E.M.

1986-01-01

To understand the mechanism of stimulus-response coupling in platelets, the potentiating effect of succinate and lithium on platelet activation was examined. The action of succinate was immediate; preincubation with succinate did not lead to desensitization. Succinate was comparable to ADP in lowering cAMP levels previously elevated by PGl 2 . Since inhibition of cAMP is not a prerequisite for platelet activation, the mechanism of potentiation of succinate remains undefined. Lithium has also been shown to inhibit adenylate cyclase in PGl 2 -pretreated platelets. Lithium, however, can also inhibit inositol phosphate (InsP) phosphatase and lead to an accumulation of InsP. In human platelets, lithium also enhanced the thrombin-induced accumulation of [ 3 H]inositol-labelled inositol trisphosphate (InsP 3 ), and inositol bisphosphate (InsP 2 ). One hour after thrombin addition, all 3 inositol phosphates returned to near basal levels. In the presence of lithium, while labelled InsP 2 and InsP 3 returned to their respective basal levels, the InsP level remained elevated, consistent with the known inhibitory effect of lithium on InsP phosphatase. In thrombin-stimulated platelets prelabeled with [ 32 P]phosphate, lithium led to a decrease in labelled phosphatidylinositol 4-phosphate (PtdIns4P) as well as an enhanced production of labelled lysophosphatidylinositol, suggesting multiple effects of lithium on platelet phosphoinositide metabolism. These observed effects, however, occurred too slowly to be the mechanism by which lithium potentiated agonist-induced platelet activation. To study the agonist-receptor interaction, the effect of the specific, high affinity thrombin inhibitor, hirudin, on thrombin-induced accumulation of [ 3 H]inositol-labelled inositol phosphates was studied

Dissociation of SERPINE1 mRNA from the translational repressor proteins Ago2 and TIA-1 upon platelet activation.

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Corduan, Aurélie; Plé, Hélène; Laffont, Benoit; Wallon, Thérèse; Plante, Isabelle; Landry, Patricia; Provost, Patrick

2015-05-01

Platelets play an important role in haemostasis, as well as in thrombosis and coagulation processes. They harbour a wide variety of messenger RNAs (mRNAs), that can template de novo protein synthesis, and an abundant array of microRNAs, which are known to mediate mRNA translational repression through proteins of the Argonaute (Ago) family. The relationship between platelet microRNAs and proteins capable of mediating translational repression, however, remains unclear. Here, we report that half of platelet microRNAs is associated to mRNA-regulatory Ago2 protein complexes, in various proportions. Associated to these Ago2 complexes are platelet mRNAs known to support de novo protein synthesis. Reporter gene activity assays confirmed the capacity of the platelet microRNAs, found to be associated to Ago2 complexes, to regulate translation of these platelet mRNAs through their 3'UTR. Neither the microRNA repertoire nor the microRNA composition of Ago2 complexes of human platelets changed upon activation with thrombin. However, under conditions favoring de novo synthesis of Plasminogen Activator Inhibitor-1 (PAI-1) protein, we documented a rapid dissociation of the encoding platelet SERPINE1 mRNA from Ago2 protein complexes as well as from the translational repressor protein T-cell-restricted intracellular antigen-1 (TIA-1). These findings are consistent with a scenario by which lifting of the repressive effects of Ago2 and TIA-1 protein complexes, involving a rearrangement of proteinmRNA complexes rather than disassembly of Ago2microRNA complexes, would allow translation of SERPINE1 mRNA into PAI-1 in response to platelet activation.

Clinical-grade quality platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelet concentrates promoted expansion of mesenchymal stromal cells.

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Borghese, C; Agostini, F; Durante, C; Colombatti, A; Mazzucato, M; Aldinucci, D

2016-08-01

The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelets as a source of growth factors for the expansion of mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl2 -activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs. © 2016 International Society of Blood Transfusion.

Reproducibility of Manual Platelet Estimation Following Automated Low Platelet Counts

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Zainab S Al-Hosni

2016-11-01

Full Text Available Objectives: Manual platelet estimation is one of the methods used when automated platelet estimates are very low. However, the reproducibility of manual platelet estimation has not been adequately studied. We sought to assess the reproducibility of manual platelet estimation following automated low platelet counts and to evaluate the impact of the level of experience of the person counting on the reproducibility of manual platelet estimates. Methods: In this cross-sectional study, peripheral blood films of patients with platelet counts less than 100 × 109/L were retrieved and given to four raters to perform manual platelet estimation independently using a predefined method (average of platelet counts in 10 fields using 100× objective multiplied by 20. Data were analyzed using intraclass correlation coefficient (ICC as a method of reproducibility assessment. Results: The ICC across the four raters was 0.840, indicating excellent agreement. The median difference of the two most experienced raters was 0 (range: -64 to 78. The level of platelet estimate by the least-experienced rater predicted the disagreement (p = 0.037. When assessing the difference between pairs of raters, there was no significant difference in the ICC (p = 0.420. Conclusions: The agreement between different raters using manual platelet estimation was excellent. Further confirmation is necessary, with a prospective study using a gold standard method of platelet counts.

The role of platelets during reproduction.

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Isermann, Berend; Nawroth, Peter P

2006-01-01

The availability of mice with defined defects within the hemostatic system enabled researchers to identify a role the coagulation system for embryonic and placental development. However, the role of platelets during development has only recently been experimentally addressed, giving some insight into potential functions of platelets during development. Thus, a quantitative embryonic platelet defect (severe thrombopenia secondary to NF-E2 deficiency) is associated with an embryonic growth retardation and reduced vascularisation of the placenta. Maternal platelet deficiency is associated with placental hemorrhage, which, however, does not impair embryonic or maternal survival. In vitro studies established that platelets or platelet conditioned medium regulate the invasive properties of human extravillous trophoblast cells and induce a phenotypical switch of trophoblast cells. These data imply that platelets are of relevance during placentation. Conversely, platelets and the formation of platelet-fibrin aggregates are dispensable for the development of the embryo proper, establishing that the lethal phenotypes observed in some embryo slacking coagulation regulators does not result from an inability to form platelet-fibrin aggregates, but likely reflects altered protease dependent signaling during vascular development.

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

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Wang, Su Bin; Jang, Ji Yong; Chae, Yun Hee; Min, Ji Hyun; Baek, Jin Young; Kim, Myunghee; Park, Yunjeong; Hwang, Gwi Seo; Ryu, Jae-Sang; Chang, Tong-Shin

2015-06-01

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

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Żmigrodzka, M; Guzera, M; Winnicka, A

2016-01-01

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Negative feedback regulation of human platelets via autocrine activation of the platelet-derived growth factor alpha-receptor.

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Vassbotn, F S; Havnen, O K; Heldin, C H; Holmsen, H

1994-05-13

Human platelets contain platelet-derived growth factor (PDGF) in their alpha-granules which is released during platelet exocytosis. We show by immunoprecipitation and 125I-PDGF binding experiments that human platelets have functionally active PDGF alpha-receptors, but not beta-receptors. The PDGF alpha-receptor (PDGFR-alpha) was identified as a 170-kDa glycosylated protein-tyrosine kinase as found in other cell types. Stimulation of platelets with 0.1 unit/ml thrombin resulted in a significant increase (2-5-fold) of the tyrosine phosphorylation of the PDGFR-alpha, as determined by immunoprecipitation with phosphotyrosine antiserum as well as with PDGFR-alpha antiserum. The observed thrombin-induced autophosphorylation of the PDGFR-alpha was inhibited by the addition of a neutralizing monoclonal PDGF antibody. Thus, our results suggest that the platelet PDGFR-alpha is stimulated in an autocrine manner by PDGF secreted during platelet activation. Preincubation of platelets with PDGF inhibited thrombin-induced platelet aggregation and secretion of ATP + ADP and beta-hexosaminidase. Thrombin-induced platelet aggregation was also reversed when PDGF was added 30 s after thrombin stimulation. Inhibition of the autocrine PDGF pathway during platelet activation by the PDGF antibody led to a potentiation of thrombin-induced beta-hexosaminidase secretion. Thus, the PDGFR-alpha takes part in a negative feedback regulation during platelet activation. Our demonstration of PDGF alpha-receptors on human platelets and its inhibitory function during platelet activation identifies a new possible role of PDGF in the regulation of thrombosis.

Further studies on the relationship between platelet buoyant density and platelet age

International Nuclear Information System (INIS)

Boneu, B.; Vigoni, F.; Boneu, A.; Caranobe, C.; Sie, P.

1982-01-01

The relationship between platelet buoyant density and platelet age was investigated in eight human subjects submitted to an autologous chromium labeled platelet survival study. Platelets were isolated after isopycnic centrifugation using eight discontinuous isoosmotic stractan gradients (five subjects), or various continuous and linear isoosmolar gradients (three subjects). A paradoxical radioactivity enrichment of the dense platelets and a premature loss of radioactivity in the light platelets were observed. These results are explained by a shift of the radioactivity distribution curve toward higher densities during the 3-4 days after platelet injection, while the standard deviation of the distribution was conserved throughout the platelet life span. These results suggest that young platelets are heterogeneous and slightly less dense than the total platelet population

[Thrombopenia and radial aplasia: 2 cases with platelet function and ultrastructural studies of megakaryocytes and platelets (author's transl)].

Science.gov (United States)

Juhan, I; Bayle, J; Mattei, J F; Thevenieau, D; Perrimond, H; Muratore, R

1979-10-01

The authors report on two cases of congenital thrombopenia with radial aplasia. Both children display several formative abnormalities and a mild thrombopenia; hemorragic manifestations occurred in the first case only. Megacryoblastic to platelets series, as studied with electronic microscopy, show small-sized, "microcytic" and hypogranular megacaryocytes, displaying a maturative disorder (dysmegacaryocytopoiesis). In functional studies, platelets of the first patient show an imperfect nucleotidic release and do not agregate normally with ristocetin. The second case exhibits mostly a PF3 reduction. The variety of expression of the megacaryocytic-platelets disorders appears likewise in the squelettal and visceral malformations. The whole disorder could be ascribed to a pleiotropic abnormal gene with a variable expressivity.

Model-based analysis of thromboxane B2 and prostaglandin E2 as biomarkers in the safety evaluation of naproxen

International Nuclear Information System (INIS)

Sahota, Tarjinder; Sanderson, Ian; Danhof, Meindert; Della Pasqua, Oscar

2014-01-01

The assessment of safety in traditional toxicology protocols relies on evidence arising from observed adverse events (AEs) in animals and on establishing their correlation with different measures of drug exposure (e.g., C max and AUC). Such correlations, however, ignore the role of biomarkers, which can provide further insight into the underlying pharmacological mechanisms. Here we use naproxen as a paradigm drug to explore the feasibility of a biomarker-guided approach for the prediction of AEs in humans. A standard toxicology protocol was set up for the evaluation of effects of naproxen in rat, in which four doses were tested (7.5, 15, 40 and 80 mg/kg). In addition to sparse blood sampling for the assessment of exposure, thromboxane B 2 and prostaglandin E 2 were also collected in satellite groups. Nonlinear mixed effects modelling was used to evaluate the predictive performance of the approach. A one-compartmental model with first order absorption was found to best describe the pharmacokinetics of naproxen. A nonlinear relationship between dose and bioavailability was observed which leads to a less than proportional increase in naproxen concentrations with increasing doses. The pharmacodynamics of TXB 2 and PGE 2 was described by direct inhibition models with maximum pharmacological effects achieved at doses > 7.5 mg/kg. The predicted PKPD relationship in humans was within 10-fold of the values previously published. Moreover, our results indicate that biomarkers can be used to assess interspecies differences in PKPD and extrapolated data from animals to humans. Biomarker sampling should be used systematically in general toxicity studies. - Highlights: • Prediction of a drug's safety profile from preclinical protocols remains challenging. • Pharmacokinetic measures of safe exposure (e.g., AUC) ignore the role of biomarkers. • PKPD relationships enable the evaluation of adverse events in a mechanistic manner. • Major differences exist between rats and

Measurement of 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid and its metabolite 12-oxo-5Z,8E,10E-heptadecatrienoic acid in human plasma by gas chromatography/negative ion chemical ionization mass spectrometry

International Nuclear Information System (INIS)

Hofmann, U.; Seefried, S.; Meese, C.O.; Mettang, T.; Huebel, E.K.; Kuhlmann, U.

1990-01-01

Thromboxane A2, the predominant product of arachidonic acid metabolism in the blood platelet, is a potent vasoconstrictor and platelet agonist. During its biosynthesis from cyclic endoperoxide, 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) is formed in equal amounts. The further metabolism of HHT, catalyzed by 15-hydroxyprostaglandin dehydrogenase, leads to 12-oxo-5Z,8E,10E-heptadecatrienoic acid (Oxo-HT). Sample workup procedures are described which allow for the sensitive and reproducible determination of these two arachidonic acid metabolites in human plasma by gas chromatography-mass spectrometry in the presence of deuterated analogues as internal standards. HHT is derivatized to the pentafluorobenzyl ester tert-butyldimethylsilyl ether. In order to enable quantification of low concentrations of about 10 pg/ml in nonstimulated human plasma, the samples have to be purified by HPLC. Oxo-HT is derivatized to the pentafluorobenzyl ester, which is purified by HPLC, and then derivatized to the trimethylsilyloxime. The method allows quantification of Oxo-HT in concentrations down to 10 pg/ml plasma. The reported methods have been used to measure HHT and Oxo-HT in stimulated platelet rich plasma and to quantify HHT in nonstimulated plasma. Determination of endogenous levels of these two arachidonic acid metabolites may give new insights into the overall biosynthesis of thromboxane A2 in man

Preparation of a viable population of indium-111-labelled human blood platelets

International Nuclear Information System (INIS)

Heyns, A.; Badenhorst, P.N.; Pieters, H.; Loetter, M.G.; Minnaar, P.C.; Duyvene de Wit, L.J.; Reenen, O.R. van; Retief, F.P.; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein; University of the Orange Free State, Bloemfontein

1980-01-01

Factors influencing labelling of human platelets with 111 Indium-8-hydroxyquinoline ([ 111 In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22 0 C with [ 111 In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally suspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubationin PPP at 37 0 C for 30 min. The 111 In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies. (orig.) [de

Demonstration of a specific C3a receptor on guinea pig platelets

International Nuclear Information System (INIS)

Fukuoka, Y.; Hugli, T.E.

1988-01-01

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin

Active Oxygen Metabolites and Thromboxane in Phorbol Myristate Acetate Toxicity to the Isolated, Perfused Rat Lung.

Science.gov (United States)

Carpenter, Laurie Jean

When administered intravenously or intratracheally to rats, rabbits and sheep, phorbol myristate acetate (PMA) produces changes in lung morphology and function are similar to those seen in humans with the adult respiratory distress syndrome (ARDS). Therefore, it is thought that information about the mechanism of ARDS development can be gained from experiments using PMA-treated animals. Currently, the mechanisms by which PMA causes pneumotoxicity are unknown. Results from other studies in rabbits and in isolated, perfused rabbit lungs suggest that PMA-induced lung injury is mediated by active oxygen species from neutrophils (PMN), whereas studies in sheep and rats suggest that PMN are not required for the toxic response. The role of PMN, active oxygen metabolites and thromboxane (TxA_2) in PMA-induced injury to isolated, perfused rat lungs (IPLs) was examined in this thesis. To determine whether PMN were required for PMA to produce toxicity to the IPL, lungs were perfused for 30 min with buffer containing various concentrations of PMA (in the presence or absence of PMN). When concentrations >=q57 ng/ml were added to medium devoid of added PMN, perfusion pressure and lung weight increased. When a concentration of PMA (14-28 ng/ml) that did not by itself cause lungs to accumulate fluid was added to the perfusion medium containing PMN (1 x 10 ^8), perfusion pressure increased, and lungs accumulated fluid. These results indicate that high concentrations of PMA produce lung injury which is independent of PMN, whereas injury induced by lower concentrations is PMN-dependent. To examine whether active oxygen species were involved in mediating lung injury induced by PMA and PMN, lungs were coperfused with the oxygen radical scavengers SOD and/or catalase. Coperfusion with either or both of these enzymes totally protected lungs against injury caused by PMN and PMA. These results suggest that active oxygen species (the hydroxyl radical in particular), mediate lung injury in

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

Science.gov (United States)

Hermann, Martin; Nussbaumer, Oliver; Knöfler, Ralf; Hengster, Paul; Nussbaumer, Walter; Streif, Werner

2010-01-01

BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

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

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

2011-08-18

Insomnia is a common sleep disorder frequently occurring in chronic alcoholic patients. Neurobiological basis of insomnia, as well as of alcoholism, is associated with disrupted functions of the main neurotransmitter systems, including the serotonin (5-hydroxytryptamine, 5-HT) system. Blood platelets are considered a limited peripheral model for the central 5-HT neurons, since both platelets and central 5-HT synaptosomes have similar dynamics of 5-HT. Platelet 5-HT concentration and platelet monoamine oxidase type B (MAO-B) are assumed to represent biomarkers for particular symptoms and behaviors in psychiatric disorders. The hypothesis of this study was that platelet 5-HT concentration and platelet MAO-B activity will be altered in chronic alcoholic patients with insomnia compared to comparable values in patients without insomnia. The study included 498 subjects: 395 male and 103 female medication-free patients with alcohol dependence and 502 healthy control subjects: 325 men and 177 women. The effects of early, middle and late insomnia (evaluated using the Hamilton Depression Rating Scale), as well as sex, age and smoking on platelet 5-HT concentration and platelet MAO-B activity were evaluated using one-way ANOVA and multiple regression analysis by the stepwise method. Platelet 5-HT concentration, but not platelet MAO-B activity, was significantly reduced in alcoholic patients with insomnia compared to patients without insomnia. Multiple regression analysis revealed that platelet 5-HT concentration was affected by middle insomnia, smoking and sex, while platelet MAO activity was affected only by sex and age. The present and previous data suggest that platelet 5-HT concentration might be used, after controlling for sex and smoking, as a biomarker for insomnia in alcoholism, PTSD and in rotating shift workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

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Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

2016-06-03

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

International Nuclear Information System (INIS)

Kawaguchi, Manami; Kitajima, Kenji; Kanokoda, Mai; Suzuki, Hidenori; Miyashita, Kazuya; Nakajima, Marino; Nuriya, Hideko; Kasahara, Kohji; Hara, Takahiko

2016-01-01

Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit − Tie2 − CD41 + Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41 + CD42b + CD61 + platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit − Tie2 − CD41 + . •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

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

Science.gov (United States)

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

2015-01-01

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