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

Aspirin affects lifespan and phagocytic uptake of platelets (A), Platelet count in control as well as aspirin-administered mice on different days.

<p>(B), Proportion of biotinylated platelets (%) in peripheral blood sample drawn from ethanol (vehicle) or aspirin (10 and 15 mg/kg) pre-administered mice 0, 24, 48, 72, and 96 h after administration of NHS-biotin. t_1/2 (h) represents platelet half-life in hours. (C) and (D), phagocytic uptake of platelets by autologous macrophages. Flow cytometry (C) and epifluorescence microscopy (D) of macrophages co-incubated with calcein-labeled platelets pretreated either with aspirin (5 mM) or ethanol (control). Scale bars, 10 µm. Data are representative of five different experiments.</p

Study of apoptosis-like features in platelets following aspirin treatment.

<p>Mitochondrial transmembrane potential (red/green ratio) (A), PS exposure (PE-annexin V binding) (B) and ROS generation (C) were studied in control platelets, as well as in cells pre-treated with aspirin as indicated. In (A), CCCP (mitochondrial protonophore) has been employed as the positive control. Data are representative of five different experiments and expressed as mean±SD. (*p<0.05 as compared to ethanol-pretreated resting platelets).</p

Study of proteosome and caspase-3 activities in aspirin-treated platelets (A), Western blots showing expression level of active Bax in platelets pretreated with ethanol, aspirin, ABT737, epoxomicin, PSI and bortezomib, as indicated (upper panel) normalized against β-actin (lower panel).

<p>(B), Quantitative representation of active Bax levels in platelet whole cell lysates determined by densitometry of Western blots. (C), caspase-3 activity from the extent of cleavage of fluorigenic substrate AC-DEVD-AMC. (D), Assay of proteasome enzymatic activity in platelets pretreated with ethanol, PSI (proteasome inhibitor) (10 µM) and aspirin. Data are representative of five different experiments and expressed as mean±SD. (*p<0.05 as compared to ethanol-pretreated resting platelets).</p

Plasma fibrinogen is a natural deterrent to amyloid β-induced platelet activation and neuronal toxicity

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, characterized by extensive loss of neurons and deposition of amyloid β (Aβ) in the form of extracellular plaques. Aβ is considered to have a critical role in synaptic loss and neuronal death underlying cognitive decline. Platelets contribute to 95% of circulating amyloid precursor protein that releases Aβ into circulation. We have recently demonstrated that the Aβ active fragment containing amino acid sequence 25–35 (Aβ<SUB>25–35</SUB>) is highly thrombogenic in nature and elicits strong aggregation of washed human platelets in a RhoA-dependent manner. In this study, we evaluated the influence of fibrinogen on Aβ-induced platelet activation. Intriguingly, Aβ failed to induce aggregation of platelets suspended in plasma but not in buffer. Fibrinogen brought about dose-dependent decline in aggregatory response of washed human platelets elicited by Aβ<SUB>25–35</SUB>, which could be reversed by increasing doses of Aβ. Fibrinogen also attenuated Aβ-induced platelet responses such as secretion, clot retraction, rise in cytosolic Ca<SUP>+2</SUP> and reactive oxygen species. Fibrinogen prevented intracellular accumulation of full-length Aβ peptide (Aβ<SUB>42</SUB>) in platelets as well as neuronal cells. We conclude that fibrinogen serves as a physiological check against the adverse effects of Aβ by preventing its interaction with cells