Biology subjects

Allan, H. E.

Publications and source records attributed to Allan, H. E..

3 recordsLinked to original sources

bioRxiv2021

Temporal in vivo platelet labelling in mice reveals age-dependent receptor expression and conservation of specific mRNAs

The proportion of young platelets, also known as newly formed or reticulated, within the overall platelet population has been clinically correlated with adverse cardiovascular outcomes. Our understanding of this is incomplete, however, because of limitations in the technical approaches available to study platelets of different ages. In this study we have developed and validated an in vivo temporal labelling approach using injectable fluorescent anti-platelet antibodies to sub-divide platelets by age and assess differences in functional and molecular characteristics. With this approach we found that young platelets (<24h old) in comparison to older platelets respond to stimuli with greater calcium flux and degranulation, and contribute more to the formation of thrombi in vitro and in vivo. Sequential sampling confirmed this altered functionality to be independent of platelet size with no size differences or changes relative to the global population seen at any age. The age associated decrease in thrombotic function was accompanied by significant decreases in the surface expression of GPVI and CD31 (PECAM-1) and an increase in CD9. Platelet mRNA content also decreased with age but at different rates for individual mRNAs indicating apparent conservation of those encoding granule proteins. Our pulse-chase type approach to define circulating platelet age has allowed timely re-examination of commonly held beliefs regarding size and reactivity of young platelets whilst providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform on age-based platelet heterogeneity in physiology and disease.

physiology↗

bioRxiv2021

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet pro-coagulant activity

BackgroundThrombosis of the lung micro-vasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting this activity. As TMEM16F plays an important role in the stimulation of the pro-coagulant activity of platelets, and considering that platelet abnormalities are common in COVID-19 patients, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. MethodsWe produced SARS-CoV-2 Spike or VSV-G protein-pseudotyped virions, or generated cells expressing Spike on their plasma membrane, and tested their effects on platelet adhesion (fluorescence), aggregation (absorbance), exposure of phosphatidylserine (flow cytometry for annexin V binding), calcium flux (flow cytometry for fluo-4 AM), and clot formation and retraction. These experiments were also conducted in the presence of the TMEM16F activity inhibitors Niclosamide and Clofazimine. ResultsHere we show that exposure to SARS-CoV-2 Spike promotes platelet activation, adhesion and spreading, both when present on the envelope of virions or upon expression on the plasma membrane of cells. Spike was effective both as a sole agonist or by enhancing the effect of known platelet activators, such as collagen and collagen-related peptide. In particular, Spike exerted a noticeable effect on the procoagulant phenotype of platelets, by enhancing calcium flux, phosphatidylserine externalisation, and thrombin generation. Eventually, this resulted in a striking increase in thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine almost abolished this Spike-induced pro-coagulant response. ConclusionsTogether, these findings provide a pathogenic mechanism to explain thrombosis associated to COVID-19 lung disease, by which Spike present in SARS-CoV-2 virions or exposed on the surface of infected cells, leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing drugs targeting this protein, such as Niclosamide, for COVID-19 therapy.

pathology↗

bioRxiv2021

Proteome and functional decline as platelets age in the circulation

Anucleate platelets circulate in the blood of healthy individuals for approximately 7-10 days during which time their protein composition may change. We hypothesized such changes would be linked to altered structure and function. Here, we separated platelets of different ages based on mRNA content and characterised them using proteomics, immunofluorescence and functional assays. Total protein content was 45{+/-}5% (n=4) lower in old platelets compared to young platelets. Predictive proteomic pathway analysis identified associations with 28 biological processes, notably increased haemostasis in young platelets and apoptosis in old platelets. Further studies confirmed platelet ageing was linked to a reduction decrease in cytoskeletal proteins, a reduction in mitochondria number, and lower calcium dynamics and granule secretion. This work delineates physical and functional changes in platelets as they age and serves as a base to examine differences associated with altered mean age of platelet populations in conditions such as immune thrombocytopenia and diabetes.

cell biology↗