Olive oil and haemostasis

Olive oil is a key component of the traditional Mediterranean diet; a diet that may explain the low rate of cardiovascular disease (CVD) in Southern European. (Extra virgin) Olive oil is a good source of monounsaturated fatty acids (MUFA) and phenolic compounds, both of which have been investigated for their effects on plasma lipids and lipoproteins, measures of oxidation and factors related to thrombosis. This issue aims to summarise the current understanding of the effects of such dietary components on the haemostatic system and subsequent risk of CVD. To date, evidence suggests that diets rich in MUFA and thus in olive oil attenuate the thrombotic response via a reduction in platelet aggregation and in postprandial FVII levels. Thrombosis is a key event in causing heart attacks and strokes, which if modulated by diet could pose a cost-effective way of reducing CVD incidence in populations that adhere to MUFA/olive oil-rich diets long-term.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis

Dietary isoflavones are currently receiving much attention because of their potential role in preventing coronary artery disease and other chronic diseases. Accumulating evidence from cell culture and laboratory animal experiments indicates that isoflavones have the potential to prevent or delay atherogenesis. Suggested mechanisms of action include: a reduction in low-density lipoprotein (LDL) cholesterol and a potential reduction in the susceptibility of the LDL particle to oxidation; (2) an improvement in vascular reactivity; (3) an inhibition of pro-inflammatory cytokines, cell adhesion proteins and nitric oxide (NO) production; and (4) an inhibition of platelet aggregation. These mechanisms are consistent with the epidemiological evidence that a high consumption of isoflavone-rich soy products is associated with a reduced incidence of coronary artery disease. Biological effects of isoflavones are dependent on many factors, including dose consumed, duration of use, protein-binding affinity, and an individual's metabolism or intrinsic oestrogenic state. Further clinical studies are necessary to determine the potential health effects of isoflavones in specific population groups as we currently know little about age-related differences in exposure to these compounds and there are few guidelines on optimal dose for cardiovascular health benefits.

Fruit polyphenols and CVD risk: a review of human intervention studies.

Epidemiological evidence suggests that polyphenols may, in part, explain the cardioprotective properties of fruits. This review aims to summarise the evidence for the effects of fruit polyphenols on four risk factors of CVD: platelet function, blood pressure, vascular function and blood lipids. This review includes human dietary intervention studies investigating fruits and their polyphenols. There was some evidence to suggest that fruits containing relatively high concentrations of flavonols, anthocyanins and procyanindins, such as pomegranate, purple grapes and berries, were effective at reducing CVD risk factors, particularly with respect to anti-hypertensive effects, inhibition of platelet aggregation and increasing endothelial-dependent vasodilation than other fruits investigated. Flavanone-rich fruits, such as oranges and grapefruits, were reported to have hypocholesterolaemic effects, with little impact on other risk factors being examined. However, the evidence was limited, inconsistent and often inconclusive. This is in part due to the heterogeneity in the design of studies, the lack of controls, the relatively short intervention periods and low power in several studies. Details of the polyphenol content of the fruits investigated were also omitted in some studies, negating comparison of data. It is recommended that large, well-powered, long-term human dietary intervention studies investigating a wider range of fruits are required to confirm these observations. Investigations into the potential synergistic effects of polyphenols on a combination of CVD risk markers, dose–response relationships and standardisation in methodology would facilitate the comparison of studies and also provide valuable information on the types of fruits which could confer protection against CVD.

LXR as a novel antithrombotic target

Liver X receptors (LXRs) are transcription factors involved in the regulation of cholesterol homeostasis. LXR ligands have athero-protective properties independent of their effects on cholesterol metabolism. Platelets are involved in the initiation of atherosclerosis and despite being anucleate express nuclear receptors. We hypothesized that the athero-protective effects of LXR ligands could be in part mediated through platelets and therefore explored the potential role of LXR in platelets. Our results show that LXR-β is present in human platelets and the LXR ligands, GW3965 and T0901317, modulated nongenomically platelet aggregation stimulated by a range of agonists. GW3965 caused LXR to associate with signaling components proximal to the collagen receptor, GPVI, suggesting a potential mechanism of LXR action in platelets that leads to diminished platelet responses. Activation of platelets at sites of atherosclerotic lesions results in thrombosis preceding myocardial infarction and stroke. Using an in vivo model of thrombosis in mice, we show that GW3965 has antithrombotic effects, reducing the size and the stability of thrombi. The athero-protective effects of GW3965, together with its novel antiplatelet/thrombotic effects, indicate LXR as a potential target for prevention of athero-thrombotic disease.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).

Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets.

Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kbeta and PI3Kgamma are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kbeta or PI3Kgamma. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein-coupled receptors for adenosine 5'-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kbeta, but not PI3Kgamma, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)-bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kbeta was a major essential regulator of platelet adhesion to fibrinogen and of integrin alpha(IIb)beta(3)-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kbeta in signaling through GPVI and integrin alpha(IIb)beta(3).

The effects of dietary nitrate on blood pressure and endothelial function: a review of human intervention studies.

Evidence has accumulated in recent years that suggests that nitrate from the diet, particularly vegetables, is capable of producing bioactive NO in the vasculature, following bioconversion to nitrite by oral bacteria. The aim of the present review was to consider the current body of evidence for potential beneficial effects of dietary nitrate on blood pressure and endothelial function, with emphasis on evidence from acute and chronic human intervention studies. The studies to date suggest that dietary nitrate acutely lowers blood pressure in healthy humans. An inverse relationship was seen between dose of nitrate consumed and corresponding systolic blood pressure reduction, with doses of nitrate as low as 3 mmol of nitrate reducing systolic blood pressure by 3 mmHg. Moreover, the current studies provide some promising evidence on the beneficial effects of dietary nitrate on endothelial function. In vitro studies suggest a number of potential mechanisms by which dietary nitrate and its sequential reduction to NO may reduce blood pressure and improve endothelial function, such as: acting as a substrate for endothelial NO synthase; increasing vasodilation; inhibiting mitochondrial reactive oxygen species production and platelet aggregation. In conclusion, the evidence for beneficial effects of dietary nitrate on blood pressure and endothelial function is promising. Further long-term randomised controlled human intervention studies assessing the potential effects of dietary nitrate on blood pressure and endothelial function are needed, particularly in individuals with hypertension and at risk of CVD.

Discovery of novel GPVI receptor antagonists by structure-based repurposing.

Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug 'resistance', underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing.

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization, secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2) activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM). Here, we demonstrate using sucrose gradient ultracentrifugation and methyl-beta-cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization, Rac1 activation, and release of ADP and thromboxane A(2) (TxA(2)). The role of ADP and TxA(2) in mediating phosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast, tyrosine phosphorylation of the GPVI-FcRgamma-chain ITAM, which has 2 YxxL motifs, is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators, and Rac activation.

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.

Clopidogrel, independent of the vascular P2Y(12) receptor, improves arterial function in small mesenteric arteries from Angll-hypertensive rats

The P2Y(12) receptor antagonist clopidogrel blocks platelet aggregation, improves systemic endothelial nitric oxide bioavailability and has anti-inflammatory effects. Since P2Y(12) receptors have been identified in the vasculature, we hypothesized that clopidogrel ameliorates Angll (angiotensin II)-induced vascular functional changes by blockade of P2Y(12) receptors in the vasculature. Male Sprague Dawley rats were infused with Angll (60 ng/min) or vehicle for 14 days. The animals were treated with clopidogrel (10 mg . kg(-1) of body weight . day(-1)) or vehicle. Vascular reactivity was evaluated in second-order mesenteric arteries. Clopidogrel treatment did not change systolic blood pressure [(mmHg) control-vehicle, 117 +/- 7.1 versus control-clopidogrel, 125 +/- 4.2; Angll vehicle, 197 +/- 10.7 versus Angll clopidogrel, 198 +/- 5.2], but it normalized increased phenylephrine-induced vascular contractions [(%KCI) vehicle-treated, 182.2 +/- 18% versus clopidogrel, 133 +/- 14%), as well as impaired vasodilation to acetylcholine [(%) vehicle-treated, 71.7 +/- 2.2 versus clopidogrel, 85.3 +/- 2.8) in Angll-treated animals. Vascular expression of P2Y(12) receptor was determined by Western blot. Pharmacological characterization of vascular P2Y(12) was performed with the P2Y(12) agonist 2-MeS-ADP [2-(methylthio) adenosine 5`-trihydrogen diphosphate trisodium]. Although 2-MeS-ADP induced endothelium-dependent relaxation [(Emax %) = 71 +/- 12%) as well as contractile vascular responses (Emax % = 83 +/- 12%), these actions are not mediated by P2Y(12) receptor activation. 2-MeS-ADP produced similar vascular responses in control and Angll rats. These results indicate potential effects of clopidogrel, such as improvement of hypertension-related vascular functional changes that are not associated with direct actions of clopidogrel in the vasculature, supporting the concept that activated platelets contribute to endothelial dysfunction, possibly via impaired nitric oxide bioavailability.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Sistema purinérgico em plaquetas de ratos adultos e interações com o sistema renina-angiotensina

As plaquetas sangüíneas são fragmentos citoplasmáticos, oriundos da ruptura dos megacariócitos, cuja principal função está relacionada à manutenção da integridade vascular. Os nucleotídeos extracelulares, ATP e ADP, bem como a adenosina, têm sido implicados em um grande número de funções fisiológicas: o ADP é o principal fator recrutador de plaquetas, enquanto que o ATP é um inibidor competitivo da agregação induzida por ADP. A adenosina é uma molécula capaz de induzir vasodilatação e inibir a agregação plaquetária. Desta maneira, a manutenção da sinalização purinérgica normal tem se mostrado importante para o tratamento de doenças cardiovasculares. Os nucleosídeos di e trifosfatos circulantes podem ser hidrolisados por membros de várias famílias de ectonucleotidases de membrana e solúveis, incluindo as ecto-nucleosídeo trifosfato difosfoidrolases (E-NTPDases) e ecto-nucleotídeo pirofosfatase/fosfodiesterases (E-NPPs), que em conjunto com a ecto-5’-nucleotidase, levam à formação de adenosina. Na superfície das plaquetas, ambas enzimas, E-NTPDase e ecto-5’-nucleotidase, estão descritas. O sistema renina-angiotensina é o principal regulador da função renal e cardiovascular, desenvolvendo um papel fundamental na homeostasia da pressão arterial e do balanço eletrolítico. A angiotensina II (ANGII) induz fisiologicamente a ativação das plaquetas, possivelmente devido às suas propriedades vasoconstritoras. Os objetivos deste trabalho foram, portanto: 1) caracterizar cineticamente a enzima E-NPP em plaquetas de ratos, utilizando o substrato marcador p-Nph-5’TMP e 2) esclarecer, mesmo que em parte, os possíveis efeitos da ANGII sobre a hidrólise extracelular de nucleotídeos por plaquetas de ratos. No primeiro capítulo deste trabalho, descrevemos uma atividade enzimática em plaquetas de ratos que compartilha as principais características bioquímicas já descritas para as E-NPPs: pH ótimo alcalino; valores de KM e Vmax calculados de aproximadamente 106.22 ± 17.83 μM e 3.44 ± 0.18 nmol p-nitrophenol/min/mg, respectivamente; e dependência de cátions divalentes. Além disso, o AMP inibiu somente a hidrólise do p-Nph-5’TMP. Por outro lado, a azida de sódio, em altas concentrações, a angiotensina II e o cloreto de gadolínio alteraram apenas as hidrólises de ATP ou ADP ou de ambos. No segundo capítulo, mostramos que a ANGII foi capaz de aumentar as hidrólises de ATP, ADP e AMP em plaquetas em todas as doses testadas (5, 50, 500 e 5000 picomóis). Entretanto, nenhuma alteração foi observada com relação à hidrólise do p-Nph-5'TMP. Em adição, observamos um aumento na hidrólise de AMP e uma diminuição na hidrólise de p-Nph-5'TMP em plaquetas de ratos espontaneamente hipertensos (SHR) quando comparados a ratos Wistar normotensos. De maneira geral, esta dissertação traz a caracterização bioquímica da enzima E-NPP na superfície de plaquetas intactas de ratos como sendo parte de um complexo sistema para a hidrólise de nucleotídeos nestes fragmentos citoplasmáticos, podendo, assim, contribuir para o desenvolvimento de terapias antiplaquetárias e para o tratamento de doenças vasculares. Adicionalmente, apresentamos alguns resultados demonstrando interações entre os sistemas angiotensinérgico e adenosinérgico de plaquetas de ratos, o que poderá contribuir para o entendimento e o tratamento de doenças cardiovasculares como hipertensão e arteriosclerose.