Platelet responses to pharmacological and physiological interventions in middle-aged men with different habitual physical activity levels.

The current guidelines following an acute coronary syndrome recommend dual-antiplatelet therapy (DAPT) (aspirin plus a P2Y12 antagonist) alongside lifestyle modifications, including more regular physical activity. It is currently unknown whether regular exercise affects the pharmacology of DAPT. AIM: To explore how exercise-induced improvements in vascular and platelet function affect the efficacy of DAPT, in a cross-sectional study of men with different physical activity levels (training status). METHODS: A total of 42 healthy, normal-weight, middle-aged men were divided into 3 groups: untrained, moderately trained and well-trained. Their platelet reactivity (agonist-induced % aggregation) was investigated in platelet-rich plasma at rest and after inhibition with aspirin and ticagrelor and/or prostacyclin and nitric oxide added to the blood in vitro, and after physiological tests of vascular function; passive movement of the leg, flow-mediated dilation and one-leg knee-extensor exercise. Vascular function of the femoral artery (changes in arterial blood flow) was assessed by ultrasound Doppler. RESULTS: Platelets from the well-trained subjects had lower basal reactivity, a higher sensitivity to the anti-aggregatory effects of prostacyclin and were more potently inhibited by DAPT compared to the untrained subjects. The moderately trained and well-trained subjects had a superior vascular function compared to untrained subjects, and their platelets were more inhibited by the passive movement, flow-mediated dilation and one-leg knee-extensor exercise. DISCUSSION: A habitually active lifestyle leads to an increased platelet sensitivity to pharmacological and physiological platelet inhibitors. We suggest that physical activity habits (training status) should be considered when personalizing and optimizing antithrombotic treatment strategies.

Inhibition of platelet aggregation ex vivo is repressed in apolipoprotein E deficient mice.

In the present study, we assessed whether the endogenous platelet inhibitory mechanisms are altered in the early to moderate stages of the atherosclerotic process. Apolipoprotein E deficient mice (ApoE-/-), a mouse model of atherosclerosis, and their wild-type (WT) counterparts were used to assess agonist-stimulated synthesis of prostacyclin (PGI2), inhibition of platelet aggregation ex vivo, and intra-platelet cAMP levels. Basal U46619 and ADP -induced platelet aggregation in vitro were increased in ApoE-/- mice at 18-20 weeks in comparison with 8-10 weeks of age. Systemically administered endothelin-1 (ET-1) or bradykinin (BK) inhibited platelet aggregation in a similar fashion in 8- to 10-week-old ApoE-/- and WT mice, but not in the ApoE-/- mice at 18-20 weeks of age, although both peptides maintained their capacity to increase plasma levels of the PGI2. Intravenous infusion of PGI2 also failed to inhibit platelet aggregation ex vivo in 18- to 20-week-old ApoE-/- mice. Interestingly, both BK and PGI2 retained their ability to increase intraplatelet cAMP in WT and ApoE-/- mice. Our results suggest that a loss of activity of endogenous inhibitorymechanisms could contribute to the increased platelet reactivity in ApoE-/- mice, and that this phenomenon occurs early in the intermediate stage of the atherosclerotic process.

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

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

Why Neighborhoods (and How We Study Them) Matter for Adolescent Development.

Adolescence is a sensitive developmental period marked by significant changes that unfold across multiple contexts. As a central context of development, neighborhoods capture-in both physical and social space-the stratification of life chances and differential distribution of resources and risks. For some youth, neighborhoods are springboards to opportunities; for others, they are snares that constrain progress and limit the ability to avoid risks. Despite abundant research on "neighborhood effects," scant attention has been paid to how neighborhoods are a product of social stratification forces that operate simultaneously to affect human development. Neighborhoods in the United States are the manifestation of three intersecting social structural cleavages: race/ethnicity, socioeconomic class, and geography. Many opportunities are allocated or denied along these three cleavages. To capture these joint processes, we advocate a "neighborhood-centered" approach to study the effects of neighborhoods on adolescent development. Using nationally representative data from the National Longitudinal Study of Adolescent to Adult Health (Add Health), we demonstrate the complex ways that these three cleavages shape specific neighborhood contexts and can result in stark differences in well-being. A neighborhood-centered approach demands more rigorous and sensitive theories of place, as well as multidimensional classification and measures. We discuss an agenda to advance the state of theories and research, drawing explicit attention to the stratifying forces that bring about distinct neighborhood types that shape developmental trajectories during adolescence and beyond.

Perceptions of African-American health professionals and community members on the participation of children and pregnant women in genetic research.

BACKGROUND: As genetic research gains more prominence in society, ethical concerns and the need for safeguards in the participation of children and pregnant women have increased. This study examined the perspectives of African-American health professional and community members on genetic research involving children and pregnant women. METHODS: We used a mixed-methods approach to collect and analyze survey data and qualitative data from focus groups of community members and structured interviews of health professionals. RESULTS: We found that community members had significantly more favorable attitudes toward participation of children and pregnant women in genetic research than health professionals. Health professionals did not differ significantly from community members in their perceived understanding of genetic research. Emergent themes included limited knowledge of genetic research and distinction of biomedical research and clinical care, ethical concerns about confidentiality and potential harm, and the need to protect children and pregnant women. Participants expressed high interest and favorable attitude towards genetic research, despite limited genetic knowledge and concerns of potential harm to children and pregnant women. Some participants felt that genetic research findings could help dispel stigma and reduce discrimination, especially in mental illness. CONCLUSION: Findings suggest that the recruitment of participants into genetic research should directly address privacy and benefit concerns, and limited knowledge of physical and mental illness genetic research. There is a critical need to invest and engage racial/ethnic communities early, provide education on genetics, mental illness, and translate and share research findings with these communities.

Aspirin has little additional anti-platelet effect in healthy volunteers receiving prasugrel.

BACKGROUND: Strong P2Y(12) blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A(2) -mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin. OBJECTIVE: The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. STUDY PARTICIPANTS/METHODS: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNow and urine samples were collected for quantification of prostanoid metabolites. RESULTS: At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose. CONCLUSIONS: In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y(12) receptor inhibitors warrants further investigation.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor.

The role of thromboxane A(2) (TxA(2)) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA(2) in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA(2) synthase (TxAS) expression and thromboxane B(2) (TxB(2)) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA(2) receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA(2) receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA(2) receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA(2) synthesis and activates its receptor in lung cancer cells. The increased TxA(2) may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

In the presence of strong P2Y12 receptor blockade, aspirin provides little additional inhibition of platelet aggregation.

BACKGROUND: Aspirin and antagonists of platelet ADP P2Y(12) receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y(12) receptors can inhibit thromboxane A(2) (TXA(2))-dependent pathways of platelet activation independently of aspirin. OBJECTIVES: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y(12) receptor blockade. METHODS: With the use of platelet-rich plasma from healthy volunteers, determinations were made in 96-well plates of platelet aggregation, TXA(2) production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP-6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1-10 µmol L(-1)), aspirin (30 µmol L(-1)), PAM + aspirin or vehicle. results: PAM concentration-dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 µmol L(-1) . In further tests of PAM (3 µmol L(-1)), aspirin (30 µmol L(-1)) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA(2) and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. CONCLUSIONS: P2Y(12) receptors are critical to the generation of irreversible aggregation through the TXA(2) -dependent pathway. As a result, strong P2Y(12) receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages.

BACKGROUND AND PURPOSE: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo. EXPERIMENTAL APPROACH: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation. KEY RESULTS: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2. CONCLUSIONS AND IMPLICATIONS: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.

Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods.

BACKGROUND: Current guidelines state that platelet aggregation studies should be conducted within 4 h of venepuncture because of the decline in sensitivity to platelet agonists. This constrains studies of platelet activity in clinical situations where samples need to be transported or there are unavoidable delays prior to assessment. OBJECTIVES: The aim of the present study was to compare systematically the responses of platelets stored in the presence of either citrate or heparin, the two most widely used anti-coagulants, using a range of standard techniques. METHODS: Blood was taken from healthy volunteers and either assessed immediately or stored at ambient temperature (18-25 degrees C) for 24 h. Platelet reactivity to a range of agonists was determined by a combination of 96-well plate techniques; light transmission aggregometry, thrombi adhesion, ATP and ADP release, and TxA(2) release; by whole blood aggregometry; and by PFA-100. RESULTS AND CONCLUSIONS: Testing using 96-well plate techniques allowed for the simultaneous measurement of responses to multiple concentrations of multiple agonists. The responses of platelets from blood anti-coagulated with heparin were predominantly preserved in all assays after 24 h storage, whereas, responses of platelets stored in blood anti-coagulated with citrate were greatly diminished. Consequently, anti-coagulation with heparin, but not citrate, preserves platelet responses for up to 24 h as determined by a range of techniques.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2.

BACKGROUND: Currently, 'aspirin resistance', the anti-platelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A(2) formation is non-linear and TXA(2) generation must be inhibited by at least 95% to inhibit TXA(2)-dependent aggregation. This relationship, however, has never been rigorously tested. OBJECTIVES: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA(2) generation and platelet activity. METHOD: Platelet aggregation, thrombi adhesion and TXA(2) production in response to arachidonic acid (0.03-1 mmol L(-1)), collagen (0.1-30 microg mL(-1)), epinephrine (0.001-100 micromol L(-1)), ADP, TRAP-6 amide and U46619 (all 0.1-30 micromol L(-1)), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. RESULTS: Platelet aggregation, adhesion and TXA(2) production induced by either arachidonic acid or collagen were inhibited in concentration-dependent manners by aspirin, with logIC(50) values that did not differ. A linear relationship existed between aggregation and TXA(2) production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R(2) 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naïve platelets, and in blood from individuals taking an anti-thrombotic dose of aspirin. CONCLUSIONS: These studies demonstrate a linear relationship between inhibition of platelet TXA(2) generation and TXA(2)-mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAID-aspirin interactions and 'aspirin resistance'.

Effects of non-steroidal anti-inflammatory drugs on cyclo-oxygenase and lipoxygenase activity in whole blood from aspirin-sensitive asthmatics vs healthy donors.

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Coronary artery brachytherapy.

Coronary artery disease is the leading cause of mortality in the West with over 1.2 million angioplasties performed annually. Despite the introduction of stents, restenosis occurs in 30-40% of vessels, which until recently has only been treated effectively by coronary artery bypass surgery. Coronary artery brachytherapy appears to provide an alternative, less invasive remedy. The mechanisms of restenosis and how these are inhibited by radiation are described here. The practicalities of radiation delivery and the history of the development of intravascular radiation as an effective clinical tool are outlined. Finally, the pitfalls of the current technology and the areas in which future research must be targeted for the field to develop are discussed.

Endothelin content, expression, and receptor type in normal and diseased human gallbladder.

The aims of this study were to characterize the endothelin (ET) system in human gallbladder by determining (1) the tissue content of ET-1 and ET-2 by ELISA; (2) the expression of mRNA of the ET precursors preproendothelin-1, -2, and -3; and (3) mRNA expression for the ETA and ETB receptors. Median content of ET-1/2 was significantly reduced in severely inflamed gallbladders compared to gallbladders with mild inflammation. There was an inverse correlation between content of ET-1/2 and inflammation score. mRNA for preproendothelin-2 was highly expressed in all samples, whereas mRNA for preproendothelin-1 was present in negligible quantities and mRNA for preproendothelin-3 was undetectable. mRNA for ETA receptors was expressed in all samples analyzed, whereas mRNA for ETB receptors was expressed at a much lower level. This study demonstrates the presence of ET-1/2 in human gallbladder. ET-1/2 content is decreased with increasing degrees of histological inflammation. ET-2 is likely to be the physiologically significant endothelin isopeptide expressed and ETA receptors appear to predominate in the human gallbladder.

Modulation of colony stimulating factor release and apoptosis in human colon cancer cells by anticancer drugs.

Modulation of the immune response against tumour cells is emerging as a valuable approach for cancer treatment. Some experimental studies have shown that secretion of colony stimulating factors by cancer cells reduces their tumorigenicity and increases their immunogenicity probably by promoting the cytolitic and antigen presenting activities of leukocytes. We have observed that human colon cancer cells (HT-29) are able to secrete granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor when stimulated with cytokines (IL-1beta and TNF-alpha). In this study we assessed, for the first time, the effects of several anticancer drugs on colony stimulating factor release or apoptosis in HT-29 cells. Cytokine-induced release of granulocyte-macrophage-colony stimulating factor, granulocyte-colony stimulating factor and macrophage-colony stimulating factor was significantly increased by cisplatin and 6-mercaptopurine. Taxol only increased macrophage-colony stimulating factor release while reduced that of granulocyte-colony stimulating factor. No changes in colony stimulating factor secretion were observed after treatment with methotrexate. Only cisplatin and taxol induced apoptosis in these cells. Secretion of colony stimulating factors by colon cancer cells may contribute to the immune host response against them. Anticancer drugs such as cisplatin and 6-mercaptopurine increase colony stimulating factor secretion by cytokine stimulated cancer cells probably through mechanisms different to those leading to cell apoptosis, an effect that may contribute to their anti-neoplasic action.

Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.