Defective diacylglycerol-induced Ca 2+ entry but normal agonist-induced activation responses in TRPC6-deficient mouse platelets. J Thromb Haemost 2012; 10: 419-29.Summary. Background: Platelet adhesion, activation and aggregation at sites of vascular injury are essential processes for primary hemostasis. Elevation of the intracellular Ca 2+ concentration is a central event in platelet activation but the underlying mechanisms are not fully understood. Store-operated calcium entry (SOCE) through Orai1 was shown to be the main Ca 2+ influx pathway in murine platelets, but there are additional non-store-operated Ca 2+ (non-SOC) and receptor operated Ca 2+ (ROC) channels expressed in the platelet plasma membrane. Objective: Canonical transient receptor potential (TRPC) channel 6 is found both in human and murine platelets and has been proposed to mediate diacylglycerol (DAG) activated ROCE but also a role in the regulation of SOCE has been suggested. Methods: To investigate the function of TRPC6 in platelet Ca 2+ signaling and activation, we analyzed platelets from mice deficient in TRPC6 using a wide range of in vitro and in vivo assays. Results: In the mutant platelets, DAG activated Ca 2+ influx was found to be abolished. However, this did not significantly affect SOCE or agonist induced Ca 2+ responses. Platelet function in vitro and in vivo was also unaltered in the absence of TRPC6. Conclusion: Our results indicate that DAG activated ROCE is mediated exclusively by TRPC6 in murine platelets, but this Ca 2+ influx has no major functional relevance for hemostasis and thrombosis. Further, in contrast to previous suggestions, based on studies with human platelets, TRPC6 appears to play an insignificant role in the regulation of SOCE in murine platelets.

Background: Exposure to air pollution increases cardiovascular morbidity and mortality. Preventing chronic cardiovascular diseases caused by air pollution relies on detecting the early effects of pollutants on the risk of cardiovascular disease development, which is limited by the lack of sensitive biomarkers. We have previously identified promising biomarkers in experimental animals but comparable evidence in humans is lacking. Methods: Air pollution is substantially worse in Beijing than in Los Angeles. We collected urine and blood samples from 26 nonsmoking, healthy adult residents of Los Angeles (mean age, 23.8 years; 14 women) before, during, and after spending 10 weeks in Beijing during the summers of 2014 and 2015. We assessed a panel of circulating biomarkers indicative of lipid peroxidation and inflammation. Personal exposure to polycyclic aromatic hydrocarbons (PAHs), a group of combustion-originated air pollutants, was assessed by urinary PAH metabolite levels. Results: Urinary concentrations of 4 PAH metabolites were 176% (95% CI, 103% to 276%) to 800% (95% CI, 509% to 1780%) greater in Beijing than in Los Angeles. Concentrations of 6 lipid peroxidation biomarkers were also increased in Beijing, among which 5-, 12-, and 15-hydroxyeicosatetraenoic acid and 9- and 13-hydroxyoctadecadienoic acid levels reached statistical significance (false discovery rate <5%), but not 8-isoprostane (20.8%; 95% CI, −5.0% to 53.6%). The antioxidative activities of paraoxonase (−9.8%; 95% CI, −14.0% to −5.3%) and arylesterase (−14.5%; 95% CI, −22.3% to −5.8%) were lower and proinflammatory C-reactive protein (101%; 95% CI, 3.3% to 291%) and fibrinogen (48.3%; 95% CI, 4.9% to 110%) concentrations were higher in Beijing. Changes in all these biomarkers were reversed, at least partially, after study participants returned to Los Angeles. Changes in most outcomes were associated with urinary PAH metabolites ( P <0.05). Conclusions: Traveling from a less-polluted to a more-polluted city induces systemic pro-oxidative and proinflammatory effects. Changes in the levels of 5-, 12-, and 15-hydroxyeicosatetraenoic acid and 9- and 13-hydroxyoctadecadienoic acid as well as paraoxonase and arylesterase activities in the blood, in association with exposures to PAH metabolites, might have important implications in preventive medicine as indicators of increased cardiovascular risk caused by air pollution exposure.

Thrombosis is a major cause of mortality in patients with myeloproliferative neoplasms (MPNs), though there is currently little to offer patients with MPN beyond aspirin and cytoreductive therapies such as hydroxyurea for primary prevention. Thrombogenesis in MPN involves multiple cellular mechanisms, including platelet activation and neutrophil-extracellular trap formation; therefore, an antithrombotic agent that targets one or more of these processes would be of therapeutic benefit in MPN. Here, we treated the JAK2V617F knockin mouse model of polycythemia vera with N-acetylcysteine (NAC), a sulfhydryl-containing compound with broad effects on glutathione replenishment, free radical scavenging, and reducing disulfide bonds, to investigate its antithrombotic effects in the context of MPN. Strikingly, NAC treatment extended the lifespan of JAK2V617F mice without impacting blood counts or splenomegaly. Using an acute pulmonary thrombosis model in vivo, we found that NAC reduced thrombus formation to a similar extent as the irreversible platelet inhibitor aspirin. In vitro analysis of platelet activation revealed that NAC reduced thrombin-induced platelet-leukocyte aggregate formation in JAK2V617F mice. Furthermore, NAC reduced neutrophil extracellular trap formation in primary human neutrophils from patients with MPN as well as healthy controls. These results provide evidence that N-acetylcysteine inhibits thrombosis in JAK2V617F mice and provide a pre-clinical rationale for investigating NAC as a therapeutic to reduce thrombotic risk in MPN.

BackgroundExposures to ambient particulate matter (PM) are associated with increased morbidity and mortality. PM2.5 (<2.5 μm) and ozone exposures have been shown to associate with carotid intima media thickness in humans. Animal studies support a causal relationship between air pollution and atherosclerosis and identified adverse PM effects on HDL functionality.We aimed to determine whether brief exposures to PM2.5 and/or ozone could induce effects on HDL anti-oxidant and anti-inflammatory capacity in humans.MethodsSubjects were exposed to fine concentrated ambient fine particles (CAP) with PM2.5 targeted at 150 μg/m3, ozone targeted at 240 μg/m3 (120 ppb), PM2.5 plus ozone targeted at similar concentrations, and filtered air (FA) for 2 h, on 4 different occasions, at least two weeks apart, in a randomized, crossover study. Blood was obtained before exposures (baseline), 1 h after and 20 h after exposures. Plasma HDL anti-oxidant/anti-inflammatory capacity and paraoxonase activity were determined. HDL anti-oxidant/anti-inflammatory capacity was assessed by a cell-free fluorescent assay and expressed in units of a HDL oxidant index (HOI). Changes in HOI (ΔHOI) were calculated as the difference in HOI from baseline to 1 h after or 20 h after exposures.ResultsThere was a trend towards bigger ΔHOI between PM2.5 and FA 1 h after exposures (p = 0.18) but not 20 h after. This trend became significant (p <0.05) when baseline HOI was lower (<1.5 or <2.0), indicating decreased HDL anti-oxidant/anti-inflammatory capacity shortly after the exposures. There were no significant effects of ozone alone or in combination with PM2.5 on the change in HOI at both time points. The change in HOI due to PM2.5 showed a positive trend with particle mass concentration (p = 0.078) and significantly associated with the slope of systolic blood pressure during exposures (p = 0.005).ConclusionsBrief exposures to concentrated PM2.5 elicited swift effects on HDL anti-oxidant/anti-inflammatory functionality, which could indicate a potential mechanism for how particulate air pollution induces harmful cardiovascular effects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12989-016-0139-3) contains supplementary material, which is available to authorized users.

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