Key Points• Hypoxia induces altered platelet proteome/reactivity, which correlates with a prothrombotic phenotype.• CAPNS1-dependent calpain activity in platelet activation cascade is associated with hypoxia-induced thrombogenesis.Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca 21 level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments. (Blood. 2014;123(8):1250-1260

Adaptation to hypobaric hypoxia is required by animals and human in several physiological and pathological situations. Hypobaric hypoxia is a pathophysiological condition triggering redox status disturbances of cell organization leading, via oxidative stress, to proteins, lipids, and DNA damage. Identifying the molecular variables playing key roles in this process would be of paramount importance to shed light on the mechanisms known to counteract the negative effects of oxygen lack. To obtain a molecular signature, changes in the plasma proteome were studied by using proteomic approach. To enrich the low-abundance proteins in human plasma, two highly abundant proteins, albumin and IgG, were first removed. By comparing the plasma proteins of high altitude natives with those of a normal control group, several proteins with a significant alteration were found. The up-regulated proteins were identified as vitamin D-binding protein, hemopexin, alpha-1–antitrypsin, haptoglobin β-chain, apolipoprotein A1, transthyretin and hemoglobin beta chain. The down-regulated proteins were transferrin, complement C3, serum amyloid, complement component 4A and plasma retinol binding protein. Among these proteins, the alterations of transthyretin and transferrin were further confirmed by ELISA and Western blotting analysis. Since all the up- and down- regulated proteins identified above are well-known inflammation inhibitors and play a positive anti-inflammatory role, these results show that there is some adaptive mechanism that sustains the inflammation balance in high altitude natives exposed to hypobaric hypoxia.

BackgroundHypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia.MethodsIn this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis.ResultsBioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia.Conclusion/SignificanceThis study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers.

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin β chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.

Exposure to high altitude induces physiological responses due to hypoxia. Lungs being at the first level to face the alterations in oxygen levels are critical to counter and balance these changes. Studies have been done analysing pulmonary proteome alterations in response to exposure to hypobaric hypoxia. However, such studies have reported the alterations at specific time points and do not reflect the gradual proteomic changes. These studies also identify the various biochemical pathways and responses induced after immediate exposure and the resolution of these effects in challenge to hypobaric hypoxia. In the present study, using 2-DE/MS approach, we attempt to resolve these shortcomings by analysing the proteome alterations in lungs in response to different durations of exposure to hypobaric hypoxia. Our study thus highlights the gradual and dynamic changes in pulmonary proteome following hypobaric hypoxia. For the first time, we also report the possible consideration of SULT1A1, as a biomarker for the diagnosis of high altitude pulmonary edema (HAPE). Higher SULT1A1 levels were observed in rats as well as in humans exposed to high altitude, when compared to sea-level controls. This study can thus form the basis for identifying biomarkers for diagnostic and prognostic purposes in responses to hypobaric hypoxia.

Objective This study explores whether the prognosis of interstitial lung disease in rheumatoid arthritis (RA-ILD) has improved over time and assesses the potential influence of drug therapy in a large multicentre UK network. Methods We analysed data from 18 UK centres on patients meeting criteria for both RA and ILD diagnosed over a 25-year period. Data included age, disease duration, outcome and cause of death. We compared all cause and respiratory mortality between RA controls and RA-ILD patients, assessing the influence of specific drugs on mortality in four quartiles based on year of diagnosis. Results A total of 290 RA-ILD patients were identified. All cause (respiratory) mortality was increased at 30% (18%) compared with controls 21% (7%) (P =0.02). Overall, prognosis improved over quartiles with median age at death rising from 63 years to 78 years (P =0.01). No effect on mortality was detected as a result of DMARD use in RA-ILD. Relative risk (RR) of death from any cause was increased among patients who had received anti-TNF therapy [2.09 (1.1–4.0)] P =0.03, while RR was lower in those treated with rituximab [0.52(0.1–2.1)] or mycophenolate [0.65 (0.2–2.0)]. Patients receiving rituximab as their first biologic had longer three (92%), five (82%) and seven year (80%) survival than those whose first biologic was an anti-TNF agent (82%, 76% and 64%, respectively) (P =0.037). Discussion This large retrospective multicentre study demonstrates survival of patients with RA-ILD has improved. This may relate to the increasing use of specific immunosuppressive and biologic agents.

Intermittent hypoxia, initially associated with adverse effects of sleep apnea, has now metamorphosed into a module for improved sports performance. The regimen followed for improved sports performance is milder intermittent hypoxic training (IHT) as compared to chronic and severe intermittent hypoxia observed in sleep apnea. Although several studies have indicated the mechanism and enough data on physiological parameters altered by IH is available, proteome perturbations remain largely unknown. Altitude induced hypobaric hypoxia is known to require acclimatization as it causes systemic redox stress and inflammation in humans. In the present study, a short IHT regimen consisting of previously reported physiologically beneficial FIO2 levels of 13.5% and 12% was administered to human subjects. These subjects were then airlifted to altitude of 3500 m and their plasma proteome along with associated redox parameters were analyzed on days 4 and 7 of high altitude stay. We observed that redox stress and associated post-translational modifications, perturbed lipid metabolism and inflammatory signaling were induced by IHT exposure at Baseline. However, this caused activation of antioxidants, energy homeostasis mechanisms and anti-inflammatory responses during subsequent high-altitude exposure. Thus, we propose IHT as a beneficial non-pharmacological intervention that benefits individuals venturing to high altitude areas.

The objective of this research was to develop a kinetic spectrophotometric method for determination of ramipril in pure form and pharmaceutical formulations. The method was based on the reaction of carboxylic acid group of the drug with a mixture of potassium iodate (KIO3) and potassium iodide (KI) in aqueous medium at room temperature. The reaction is followed spectrophotometrically by measuring the increase in absorbance at 352 nm as a function of time. The initial-rate and fixed-time methods were adopted for constructing the calibration curves. Both the calibration curves were linear in the concentration range of 10.0-70.0 microg mL(-1). The detection limits were 0.02 microg mL(-1) and 0.15-microg mL(-1) for initial rate and fixed time methods, respectively. The proposed methods are validated statistically and through recovery studies. The point and interval hypothesis tests have been performed confirming that there is no significant difference between the proposed methods and the reference method. The experimental true bias of all samples is less than +/- 2%. The methods have been successfully applied to the determination of ramipril in tablets and capsules.