Regulatory role of proteasome in determination of platelet life span

Limit of platelet life span (8-10 days) is determined by the activity of a putative "internal clock" composed of Bcl-2 family proteins, while role of other molecular players in this process remains obscure. Here, we sought to establish a central role of proteasome in platelet life span regulation. Administration of mice with inhibitors of proteasome peptidase activity induced significant thrombocytopenia. This was associated with enhanced clearance of biotin-labeled platelets from circulation and reduction in average platelet half life from 66 to 37 h. Cells pretreated in vitro with proteasome inhibitors exhibited augmented annexin V binding and drop in mitochondrial transmembrane potential indicative of apoptotic cell death and decreased platelet life span. These cells were preferentially phagocytosed by monocyte-derived macrophages, thus linking proteasome activity with platelet survival. Decisive role of proteasome in this process was underscored from enhanced expression of conformationally active Bax in platelets with attenuated proteasome activity, which was consistent with pro-apoptotic phenotype of these cells. The present study establishes a critical role of proteasome in delimiting platelet life span ostensibly through constitutive elimination of the conformationally active Bax. These findings bear potential implications in clinical settings where proteasome peptidase activities are therapeutically targeted

Sirtuin inhibition induces apoptosis-like changes in platelets and thrombocytopenia

Sirtuins are evolutionarily conserved NAD+-dependent acetyl-lysine deacetylases that belong to class III type of histone deacetylases. In humans, seven sirtuin isoforms (Sirt1 to Sirt7) have been identified. Sirtinol, a cell permeable lactone ring derived from naphthol, is a dual Sirt1/Sirt2 inhibitor of low potency whereas EX-527 is a potent and selective Sirt1 inhibitor. Here we have demonstrated that Sirt1, Sirt2 and Sirt3 are expressed in enucleate platelets. Both sirtinol or EX-527 induced apoptosis-like changes in platelets as revealed from enhanced annexin V binding, ROS production and drop in mitochondrial transmembrane potential. Above changes were associated with increased phagocytic clearance of the platelets by macrophages. Expression of acetylated p53 and the conformationally active form of Bax were found to be significantly higher in both sirtinol- as well as EX-527-treated platelets, thus implicating p53-Bax axis in apoptosis induced by sirtuin inhibitors. Administration of either sirtinol or EX-527 in mice led to reduction in both platelet count and number of reticulated platelets. Our results, for the first time, implicate sirtuins as a central player in determination of platelet aging. Since sirtuin inhibitors are being evaluated for their anti-tumor activity, this study rekindles attention to potential side effect of sirtuin inhibition in delimiting platelet life span and management of thrombosis

Thrombolytic along with anti-platelet activity of crinumin, a protein constituent of Crinum asiaticum

Several anticoagulants, anti-platelet and thrombolytic medications are used for the treatment of thrombotic disorders. Anti-coagulants and anti-platelet agents prevent the formation of blood clots but do not dissolve existing clots, whereas thrombolytic agents are able to dissolve a clot but emboli can form even after successful treatment. Thus, none of them provide a permanent and complete solution. In this regard a single molecule that could both dissolve the clot and prevent the formation of new clots would be useful in the treatment of thrombotic diseases. Crinumin, a stable and active (in many adverse conditions) serine protease, shows plasmin-like fibrinolytic activity and inhibits platelet aggregation and P-selectin exposure, as established by photography, phase contrast microscopy, whole blood optical Lumi-aggregometry and flow cytometry. Crinumin could be an efficient and inexpensive therapeutic agent for the treatment and prevention of thromboembolic diseases

Development and characterization of highly selective target-sensitive liposomes for the delivery of streptokinase: in vitro/in vivo studies

Streptokinase is one of the most commonly used thrombolytic agents for the treatment of thromboembolism. Short half-life of the streptokinase requires administration of higher dose which results in various side effects including systemic haemorrhage due to activation of systemic plasmin. To increase the selectivity of the streptokinase and hence to reduce side effects, various novel carriers have been developed. Among these carriers, liposomes have been emerged as versatile carrier. In the present study, highly selective target-sensitive liposomes were developed and evaluated by in vitro and in vivo studies. Prepared liposomes were found to release streptokinase in vitro following binding with activated platelets. Intravital microscopy studies in thrombosed murine model revealed higher accumulation of liposomes in the thrombus area. In vivo thrombolysis study was performed in the human clot inoculated rat model. Results of the study showed that target-sensitive liposomes dissolved 28.27 ± 1.56% thrombus as compared to 17.18 ± 1.23% of non-liposomal streptokinase. Further, it was also observed that target-sensitive liposomes reduced the clot dissolution time as compared to streptokinase solution. Studies concluded that developed liposomes might be pragmatic carriers for the treatment of thromboembolism

Development and characterization of site specific target sensitive liposomes for the delivery of thrombolytic agents

In recent times, search for potent and highly selective thrombolytic agents with minimal side effects has become a major area of research. The aim of the present study was to develop and characterize target sensitive (TS) liposomes encapsulating streptokinase, a thrombolytic agent. The developed TS liposomes were composed of dioleylphophatidyl ethanolamine (DOPE) and dipalmityl-c(RGDfK) (10:1 mol/mol). Dipalmityl-c(RGDfK) was synthesized using typical carbodiimide chemistry using palmitic acid and c(RGDfK), while lysine was used as a spacer. Liposomes were of 100–120 nm size. In vitro drug release study showed that nearly 40% drug of the entrapped drug was released in 12 h in the PBS (pH 7.4), however on incubation with activated platelet about 90% of drug was released within 45 min. The results suggested target sensitivity of the liposomes. Further, targeting potential was confirmed using fluorescent microscopy and flow cytometry. Clot lysis study revealed that TS liposomes could not only reduce the clot lysis time but also increase the extent of clot lysis as compared to non-liposomal streptokinase solution. In conclusion, the present liposomal formulation will target the thrombolytic agent to the activated platelets in the thrombus and hence will improve the therapeutic efficacy of the drug

Improvement of oxidative stress and immunity by melatonin: an age dependent study in golden hamster

Reactive oxygen species (ROS) have been proposed to play an important role in balancing the pro- and antioxidant homeostasis during aging. Melatonin has been suggested as an effective free radical scavenger that might have a role during the process of aging. We observed, that melatonin administration (25 μg/100 g body weight for 30 days) significantly augments the activity of anti-oxidative enzymes like superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) in the plasma, spleen and bone marrow (BM) of young (6 weeks), adult (30 weeks) and old aged (2.5 years) male golden hamster, Mesocricetus auratus. A sharp decline in generation of ROS was observed in peripheral blood mononuclear cells (PBMC) and splenocytes upon melatonin administration in different age group of hamsters. Reduction in the level of thiobarbituric acid-reactive substances (TBARS) and total nitrite and nitrate concentration as metabolites and indicators of nitric oxide (NO) in plasma, spleen and BM were observed along with night time (22:00 h) melatonin concentration in different age group of hamsters after administration of melatonin and compared to the control group (treated with 0.9% saline). General immune parameters like proliferation of splenocytes, PBMC and colony forming ability of GM-CFU were observed following melatonin treatment in different age group, although it was low only in aged hamsters compared to the young and adult. Our data indicates that the age related increase of oxidative load and simultaneously augments the general immunity in aged hamsters

Aspirin Delimits Platelet Life Span by Proteasomal Inhibition

<div><p>Aspirin is widely used in clinical settings as an anti-inflammatory and anti-platelet drug due its inhibitory effect on cyclooxygenase activity. Although the drug has long been considered to be an effective and safe therapeutic regime against inflammatory and cardiovascular disorders, consequences of its cyclooxygenase-independent attributes on platelets, the key players in thrombogenesis, beg serious investigation. In this report we explored the effect of aspirin on platelet lifespan in murine model and its possible cytotoxicity against human platelets <i>in vitro</i>. Aspirin administration in mice led to significant reduction in half-life of circulating platelets, indicative of enhanced rate of platelet clearance. Aspirin-treated human platelets were found to be phagocytosed more efficiently by macrophages, associated with attenuation in platelet proteasomal activity and upregulation of conformationally active Bax, which were consistent with enhanced platelet apoptosis. Although the dosage of aspirin administered in mice was higher than the therapeutic regimen against cardiovascular events, it is comparable with the recommended anti-inflammatory prescription. Thus, above observations provide cautionary framework to critically re-evaluate prophylactic and therapeutic dosage regime of aspirin in systemic inflammatory as well as cardiovascular ailments.</p></div