Assessment of in vitro Effects of Anthocyanins on Platelet Function.

BACKGROUND: Increased platelet activity plays a significant role in the development of arterial thrombosis and cardiovascular disease (CVD). Natural antioxidants including anthocyanin (AC) have gained considerable interest due to their hypothesized antithrombotic potential. PRIMARY STUDY OBJECTIVE: Our study aimed to examine the in vitro effect of AC compounds on platelet activation and aggregation. METHODS: Fasting blood samples were collected from healthy volunteers (n = 13). A full blood examination was done to exclude any abnormal specimen. Flow cytometer assessed platelet activity by recording platelet surface markers expression of P-selectin (CD62P) and PAC-1. Platelet aggregation studies were performed by stimulating platelets using three different agonists adenosine diphosphate (ADP), collagen and arachidonic acid (AA). SETTING: The study was done in the school of Medical Sciences, Griffith University. PARTICIPANTS: Thirteen healthy adult participants were involved for blood collection. INTERVENTION: AC was prepared using hemicellulose capsules sourced from Bilberries and Black Currants. RESULTS: Anthocyanin (50 mg/L) significantly inhibited AA-induced platelet aggregation. Expression of P-selectin was significantly suppressed by 50 mg/L AC as measured by flow cytometer. CONCLUSIONS: AC attenuates platelet function by suppressing P-selectin expression and influencing Thromboxane A2 pathway (AA stimulation). These results provide further evidence for the effect of AC and the possible mechanism by which AC reduces platelet aggregation and activation. This study supports future human intervention trials to show that AC may act as a complement to other antiplatelet agents in reducing the risk of thrombosis.

Efficient Blocking of Enterovirus 71 Infection by Heparan Sulfate Analogues Acting as Decoy Receptors.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease, for which there is no antiviral therapy. We have developed densely sulfated disaccharide heparan sulfate (HS) analogues that are potent small molecule inhibitors of EV71 infection, binding to the viral capsid and acting as decoy receptors to block early events of virus replication. The simplified structures, more potent than defined HS disaccharides and with no significant anticoagulant activity, offer promise as anti-EV71 agents.

Oxidative stress biomarkers in type 2 diabetes mellitus for assessment of cardiovascular disease risk.

AIMS: Type-2 Diabetes Mellitus (T2DM) is one of the most prevalent and progressive metabolic conditions affecting approximately 8.5% of the global population. Individuals with T2DM have a significantly increased risk of developing chronic conditions such as cardiovascular disease (CVD) and its associated complications, therefore, it is of great importance to establish strategies for combatting T2DM and its associated chronic conditions. Current literature has identified several biomarkers that are known to play a key role in the pathogenesis of CVD. Many of these biomarkers affecting CVD are influenced by an increase in oxidative stress as seen in T2DM. The purpose of this review is to analyse and correlate the oxidative stress-related biomarkers that have been identified in the literature to provide an updated summary of their significance in CVD risk factors. DATA SYNTHESIS: This review has analysed current research on T2DM, CVD, and oxidative stress. Four key cardiovascular risk factors: thrombosis, inflammation, vascular homeostasis and cellular proliferation were searched to identify potential biomarkers for this review. These biomarkers stem from seven major cellular pathways; NF-κB, Keap1-Nrf2, protein kinase-C, macrophage activation, arachidonic acid mobilisation, endothelial dysfunction and advanced glycation end products. CONCLUSIONS: The pathways and biomarkers were analysed to show their role as contributing factors to CVD development and a summary is made regarding the assessment of cardiovascular risk in T2DM individuals.

Mildly elevated unconjugated bilirubin is associated with reduced platelet activation-related thrombogenesis and inflammation in Gilbert's syndrome.

Gilbert's syndrome (GS) is associated with a mild unconjugated hyperbilirubinemia, increased circulating antioxidant capacity, and reduced cardiovascular disease (CVD) risk. The current study investigated whether mildly elevated circulating unconjugated bilirubin (UCB) is negatively associated with multiple thrombotic risk factors including platelet activity, hemostatic function, and inflammation in individuals with GS. Blood samples were collected from matched GS and control subjects (14 per group). Activation-dependent platelet surface marker expression of PAC-1 (binds to GPIIb/IIIa surface receptors on activated platelets) and CD62P (marker for P-selectin released from activated degranulated platelets) was assessed in adenosine diphosphate (ADP)-stimulated platelets using flow cytometry. Exogenous agonists, ADP, collagen, and arachidonic acid (AA), were used to stimulate platelet aggregation. A statistically significant decrease in the expression of P-selectin (P = 0.030) on activated platelets was observed in GS subjects. Collagen and AA-induced platelet aggregation were significantly (P = 0.018; P = 0.032 for respective agonists) reduced in GS versus control group. Elevated UCB (P = 0.001) and high density lipoprotein (P = 0.033) in addition to reduced low density lipoprotein (P = 0.024) and high sensitive C-reactive protein (P = 0.043) were also observed in GS when compared to the control group. Reduced P-selectin expression suggests decreased platelet activation-dependent degranulation, while reduced platelet aggregation by AA and collagen indicates a quantitative decrease in platelet aggregation consequently targeting the cyclooxygenase-1 and GP VI pathways, respectively. These findings are the first to demonstrate that the activation of platelets is mildly inhibited in individuals with GS, an effect that might contribute to protection from platelet hyperactivation-induced thrombosis and thus cardiovascular mortality in individuals with benign hyperbilirubinemia.

Bilirubin, platelet activation and heart disease: a missing link to cardiovascular protection in Gilbert's syndrome?

Gilbert's syndrome (GS) is a relatively common condition, inducing a benign, non-hemolytic, unconjugated hyperbilirubinemia. Gilbert's Syndrome is associated with mutation in the Uridine Glucuronosyl Transferase 1A1 (UGT1A1) gene promoter, reducing UGT1A1 activity, which normally conjugates bilirubin allowing its elimination from the blood. Individuals with GS demonstrate mildly elevated plasma antioxidant capacity caused by elevated levels of unconjugated bilirubin (UCB), reduced thiols and glutathione. Interestingly, the development of, and risk of mortality from, cardiovascular disease is remarkably reduced in GS individuals. An explanation for this protection may be explained by bilirubin's ability to inhibit multiple processes that induce platelet hyper-reactivity and thrombosis, thus far under-appreciated in the literature. Reactive oxygen species are produced continuously via metabolic processes and have the potential to oxidatively modify proteins and lipids within cell membranes, which may encourage the development of thrombosis and CVDs. Oxidative stress induced platelet hyper-reactivity significantly increases the risk of thrombosis, which can potentially lead to tissue infarction. Here, we discuss the possible mechanisms by which increased antioxidant status might influence platelet function and link this to cardiovascular protection in GS. In summary, this is the first article to discuss the possible role of bilirubin as an anti-thrombotic agent, which inhibits platelet activation and potentially, organ infarction, which could contribute to the reduced mortality rate in mildly hyperbilirbinemic individuals.

Unconjugated bilirubin inhibits collagen induced platelet activation.

Gilbert's syndrome (GS) individuals have mildly elevated serum unconjugated bilirubin (UCB) concentrations and are protected against the development of cardiovascular diseases (CVD). Although UCB has antioxidant properties, which could delay atherosclerotic plaque development, evidence suggests UCB might also affect haemostasis, subsequently influencing thrombus formation after atherosclerotic plaque rupture. The aim of this study was to reveal the in-vitro effect of UCB on platelet function and haemostatic factors at physiologically relevant concentrations seen in GS. Blood samples were collected from 16 healthy volunteers (mean age 25 ± 5) for full blood examination. A final concentration of approximately 35 ± 4.0 µmol/L of UCB was obtained by adding 1.25 µL of UCB stock solution to 250 µL of sample, to study its effect on platelet aggregation, coagulation and lipid profile. Collagen induced platelet aggregation was significantly inhibited in platelet rich plasma treated with UCB. Coagulation and lipid profile did not change by the in-vitro addition of UCB. These data are the first to show that mildly (but physiologically) elevated UCB inhibits platelet activity in plasma via a mechanism specifically related to collagen induced platelet activation. These findings support a novel mechanism which might further explain protection from CVD by mildly elevated levels of UCB, thus reducing the risk of thrombus formation by inhibition of collagen-induced platelet aggregation.