Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals.

Background: Assessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing "one-size-fits-all" approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. Objectives: Our aim was to compare well-used platelet reactivity assays. Methods: Blood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry (Roche Diagnostics), Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. Results: A strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry large area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid-induced aggregation, and explained substantial variance (ß = -1.735; P = 4.59 × 10-780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (ß = -1.612; P = 6.75 × 10-27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. Conclusion: Caution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

Granulocyte- Macrophage Colony-Stimulating Factor Reverses Immunosuppression Acutely Following a Traumatic Brain Injury and Hemorrhage Polytrauma in a Juvenile Male Rat Model.

Traumatic brain injury (TBI) is a common cause of morbidity and mortality in children. We have previously shown that TBI with a concurrent extracranial injury reliably leads to post-injury suppression of the innate and adaptive immune systems. In patients with post-injury immune suppression, if immune function could be preserved, this might represent a therapeutic opportunity. As such, we examined, in an animal injury model, whether systemic administration of granulocyte macrophage colony-stimulating factor (GM-CSF) could reverse post-injury immune suppression and whether treatment was associated with neuroinflammation or functional deficit. Prepubescent male rats were injured using a controlled cortical impact model and then subjected to removal of 25% blood volume (TBI/H). Sham animals underwent surgery without injury induction, and the treatment groups were sham and injured animals treated with either saline vehicle or 50 µg/kg GM-CSF. GM-CSF was administered following injury and then daily until sacrifice at post-injury day (PID) 7. Immune function was measured by assessing tumor necrosis factor-α (TNF-α) levels in whole blood and spleen following ex vivo stimulation with pokeweed mitogen (PWM). Brain samples were assessed by multiplex enzyme-linked immunosorbent assay (ELISA) for cytokine levels and by immunohistochemistry for microglia and astrocyte proliferation. Neuronal cell count was examined using cresyl violet staining. Motor coordination was evaluated using the Rotarod performance test. Treatment with GM-CSF was associated with a significantly increased response to PWM in both whole blood and spleen. GM-CSF in injured animals did not lead to increases in levels of pro-inflammatory cytokines in brain samples but was associated with significant increases in counted astrocytes. Finally, while injured animals treated with saline showed a significant impairment on behavioral testing, injured animals treated with GM-CSF performed similarly to uninjured animals. GM-CSF treatment in animals with combined injury led to increased systemic immune cell response in whole blood and spleen in the acute phase following injury. Improved immune response was not associated with elevated pro-inflammatory cytokine levels in the brain or functional impairment.

Oral nitrate supplementation improves cardiovascular risk markers in COPD: ON-BC, a randomised controlled trial.

BACKGROUND: Short-term studies suggest that dietary nitrate (NO3 -) supplementation may improve the cardiovascular risk profile, lowering blood pressure (BP) and enhancing endothelial function. It is not clear if these beneficial effects are sustained and whether they apply in people with COPD, who have a worse cardiovascular profile than those without COPD. Nitrate-rich beetroot juice (NR-BRJ) is a convenient dietary source of nitrate. METHODS: The ON-BC trial was a randomised, double-blind, placebo-controlled parallel group study in stable COPD patients with home systolic BP (SBP) measurement ≥130 mmHg. Participants were randomly allocated (1:1) using computer-generated, block randomisation to either 70 mL NR-BRJ (400 mg NO3 -) (n=40) or an otherwise identical nitrate-depleted placebo juice (0 mg NO3 -) (n=41), once daily for 12 weeks. The primary end-point was between-group change in home SBP measurement. Secondary outcomes included change in 6-min walk distance (6MWD) and measures of endothelial function (reactive hyperaemia index (RHI) and augmentation index normalised to a heart rate of 75 beats·min-1 (AIx75)) using an EndoPAT device. Plasma nitrate and platelet function were also measured. RESULTS: Compared with placebo, active treatment lowered SBP (Hodges-Lehmann treatment effect -4.5 (95% CI -5.9- -3.0) mmHg), and improved 6MWD (30.0 (95% CI 15.7-44.2) m; p<0.001), RHI (0.34 (95% CI 0.03-0.63); p=0.03) and AIx75 (-7.61% (95% CI -14.3- -0.95%); p=0.026). CONCLUSIONS: In people with COPD, prolonged dietary nitrate supplementation in the form of beetroot juice produces a sustained reduction in BP, associated with an improvement in endothelial function and exercise capacity.

Immature platelet dynamics are associated with clinical outcomes after major trauma.

BACKGROUND: Major trauma results in dramatic changes in platelet behavior. Newly formed platelets are more reactive than older platelets, but their contributions to hemostasis and thrombosis after severe injury have not been previously evaluated. OBJECTIVES: To determine how immature platelet metrics and plasma thrombopoietin relate to clinical outcomes after major injury. METHODS: A prospective observational cohort study was performed in adult trauma patients. Platelet counts and the immature platelet fraction (IPF) were measured at admission and 24 hours, 72 hours, and 7 days after injury. Thromboelastometry was performed at admission. Plasma thrombopoietin, c-Mpl, and GPIbα were quantified in a separate cohort. The primary outcome was in-hospital mortality; secondary outcomes were venous thromboembolic events and multiple organ dysfunction syndrome (MODS). RESULTS: On admission, immature platelet counts (IPCs) were significantly lower in nonsurvivors (n = 40) than in survivors (n = 236; 7.3 × 109/L vs 10.6 × 109/L; P = .009), but IPF did not differ. Similarly, impaired platelet function on thromboelastometry was associated with lower admission IPC (9.1 × 109/L vs 11.9 × 109/L; P < .001). However, at later time points, we observed significantly higher IPF and IPC in patients who developed venous thromboembolism (21.0 × 109/L vs 11.1 × 109/L; P = .02) and prolonged MODS (20.9 × 109/L vs 11 × 109/L; P = .003) than in those who did not develop complications. Plasma thrombopoietin levels at admission were significantly lower in nonsurvivors (P < .001), in patients with MODS (P < .001), and in those who developed venous thromboembolism (P = .04). CONCLUSION: Lower levels of immature platelets in the acute phase after major injury are associated with increased mortality, whereas higher immature platelet levels at later time points may predispose to thrombosis and MODS.

Renal Function Underpins the Cyclooxygenase-2: Asymmetric Dimethylarginine Axis in Mouse and Man.

Introduction: Through the production of prostacyclin, cyclooxygenase (COX)-2 protects the cardiorenal system. Asymmetric dimethylarginine (ADMA), is a biomarker of cardiovascular and renal disease. Here we determined the relationship between COX-2/prostacyclin, ADMA, and renal function in mouse and human models. Methods: We used plasma from COX-2 or prostacyclin synthase knockout mice and from a unique individual lacking COX-derived prostaglandins (PGs) because of a loss of function mutation in cytosolic phospholipase A2 (cPLA2), before and after receiving a cPLA2-replete transplanted donor kidney. ADMA, arginine, and citrulline were measured using ultra-high performance liquid-chromatography tandem mass spectrometry. ADMA and arginine were also measured by enzyme-linked immunosorbent assay (ELISA). Renal function was assessed by measuring cystatin C by ELISA. ADMA and prostacyclin release from organotypic kidney slices were also measured by ELISA. Results: Loss of COX-2 or prostacyclin synthase in mice increased plasma levels of ADMA, citrulline, arginine, and cystatin C. ADMA, citrulline, and arginine positively correlated with cystatin C. Plasma ADMA, citrulline, and cystatin C, but not arginine, were elevated in samples from the patient lacking COX/prostacyclin capacity compared to levels in healthy volunteers. Renal function, ADMA, and citrulline were returned toward normal range when the patient received a genetically normal kidney, capable of COX/prostacyclin activity; and cystatin C positively correlated with ADMA and citrulline. Levels of ADMA and prostacyclin in conditioned media of kidney slices were not altered in tissue from COX-2 knockout mice compared to wildtype controls. Conclusion: In human and mouse models, where renal function is compromised because of loss of COX-2/PGI2 signaling, ADMA levels are increased.

Assessment of Platelet Function by High-Throughput Screening Light Transmission Aggregometry: Optimul Assay.

Platelet function testing is critical in the diagnosis of bleeding disorders and allows monitoring of antiplatelet therapy. The gold standard assay, light transmission aggregometry (LTA), was developed 60 years ago and remains widely used worldwide. It requires, however, access to expensive equipment and is time-consuming, and the interpretation of results requires evaluation by an experienced investigator. It also suffers from a lack of standardization, resulting in widely variable results between laboratories. 96-well plate-based Optimul aggregometry utilizes the same principles of LTA and aims to standardize agonist concentrations with the development of 96-well plates which are precoated with 7 concentrations of each lyophilized agonist (arachidonic acid, adenosine diphosphate, collagen, epinephrine, TRAP-6 amide, and U46619) and stored at ambient room temperature (20-25 °C) for up to 12 weeks. For platelet function testing, 40 µL of platelet-rich plasma is added to each well, and the plate is placed onto a plate shaker, after which platelet aggregation is determined by changes in light absorbance. This method reduces the blood volume required and allows for in-depth platelet function analysis without specialist training, or the need to purchase expensive, dedicated equipment.

What can we learn from senescent platelets, their transcriptomes and proteomes?

Research into the natural aging process of platelets has garnered much research interest in recent years, and there have long been associations drawn between the proportion of newly formed platelets in the circulation and the risk of thrombosis. However, these observations have largely been demonstrated in patient groups in which there may be underlying systemic changes that effect platelet function. Recent advances in technology have allowed in-depth analysis of differently aged platelets isolated from the peripheral blood of healthy individuals and have demonstrated that aged platelets, often referred to as senescent platelets, undergo extensive changes in the transcriptome and proteome. Ultimately, these changes result in platelets whose functions have deteriorated such that they cannot partake in hemostatic responses to the same extent as newly formed platelets. Here, we review transcriptomic and proteomic research in platelet aging in the context of health and how this research sheds light upon alterations in platelet structure and function.

Platelets normally last within the human circulation for approximately 10 days, however there are a number of diseases in which this life span is notably reduced. People who have platelets with such reduced lifespans have higher risks of heart attacks and strokes and reduced responsiveness to standard anti-platelet drugs. Research in recent years has aimed to understand the age-related changes that occur within platelets as they circulate in the bloodstream. In this review article, the authors provide a summary of the changes that occur during the natural aging process of platelets in healthy people, highlighting reductions in the levels of RNA and proteins. Despite these general reductions in their own RNA and proteins, research has shown that as platelets circulate they take up other RNA transcripts and proteins from the bloodstream, and this too seems a hallmark of platelet aging. The authors also discuss age-related declines in various aspects of platelet function which renders them less effective participants in the formation of blood clots.

Platelet ageing: A review.

Platelet ageing is an area of research which has gained much interest in recent years. Newly formed platelets, often referred to as reticulated platelets, young platelets or immature platelets, are defined as RNA-enriched and have long been thought to be hyper-reactive. This latter view is largely rooted in associations and observations in patient groups with shortened platelet half-lives who often present with increased proportions of newly formed platelets. Evidence from such groups suggests that an increased proportion of newly formed platelets is associated with an increased risk of thrombotic events and a reduced effectiveness of standard anti-platelet therapies. Whilst research has highlighted the existence of platelet subpopulations based on function, size and age within patient groups, the common intrinsic changes which occur as platelets age within the circulation are only just being explored. By understanding the changes that occur during the natural ageing processes of platelets, we may be able to identify the triggers for alterations in platelet life span and platelet reactivity. Here we review research on platelet ageing in the context of health and disease, paying particular attention to the experimental approaches taken and the robustness of conclusions that can be drawn.

Temporal in vivo platelet labeling 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. However, our understanding of this is incomplete 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 labeling approach using injectable fluorescent antiplatelet antibodies to subdivide platelets by age and assess differences in functional and molecular characteristics. With this approach, we found that young platelets (<24 hours old) in comparison with 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 distribution of sizes of tracked platelets commensurate with the global platelet population throughout their 5-day lifespan in the circulation. 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 messenger RNA (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 reexamination of commonly held beliefs regarding size and reactivity of young platelets while providing novel insights into the temporal regulation of receptor and protein expression. Overall, future application of this validated tool will inform age-based platelet heterogeneity in physiology and disease.

Revisiting stress-corrosion cracking and hydrogen embrittlement in 7xxx-Al alloys at the near-atomic-scale.

The high-strength 7xxx series aluminium alloys can fulfil the need for light, high strength materials necessary to reduce carbon-emissions, and are extensively used in aerospace for weight reduction purposes. However, as all major high-strength materials, these alloys can be sensitive to stress-corrosion cracking (SCC) through anodic dissolution and hydrogen embrittlement (HE). Here, we study at the near-atomic-scale the intra- and inter-granular microstructure ahead and in the wake of a propagating SCC crack. Moving away from model alloys and non-industry standard tests, we perform a double cantilever beam (DCB) crack growth test on an engineering 7xxx Al-alloy. H is found segregated to planar arrays of dislocations and to grain boundaries that we can associate to the combined effects of hydrogen-enhanced localised plasticity (HELP) and hydrogen-enhanced decohesion (HEDE) mechanisms. We report on a Mg-rich amorphous hydroxide on the corroded crack surface and evidence of Mg-related diffusional processes leading to dissolution of the strengthening η-phase precipitates ahead of the crack.

A pilot study assessing the implementation of 96-well plate-based aggregometry (Optimul) in Australia.

Identification of disordered platelet function is important to guide peri-operative bleeding management as well as long term treatment and prognostic strategies in individuals with platelet bleeding disorders. Light transmission aggregometry (LTA), the current gold standard diagnostic test of platelet function is a time consuming technique almost exclusively performed in specialised laboratories and almost universally unavailable in regional centres in Australia, where there is an unmet need for access to specialised platelet function diagnostic services. 96-well plate-based aggregometry (Optimul, UK), has been utilised in research laboratories as a novel platform to investigate platelet function. We evaluated the Optimul assay at two centres in Australia, one regional and one tertiary metropolitan, to assess its feasibility as a screening test applicable to remote regional centres. Concentration-response curves were established from 45 healthy volunteers at the participating regional hospital and from 31 healthy volunteers at the tertiary institution. Optimul successfully detected anti-platelet effects in individuals taking aspirin (n=4), NSAID (n=2), clopidogrel (n=2) and dual therapy with aspirin and clopidogrel (n=1). When tested in parallel to LTA in individuals referred for the evaluation of abnormal bleeding symptoms there was overall a very good level of agreement between Optimul and LTA [Cohen's kappa (k2)=0.84], supporting its role as a useful screening tool in the assessment of platelet function. Optimul assay performance was quick and the methodology simple, requiring no specialised training or resources to be implemented at either the regional or metropolitan laboratory. Widespread implementation, particularly in regional laboratories within Australia where specialised platelet function testing is unavailable, has the potential to drastically improve the inequity of access to such services.

Neutrophil-Derived Protein S100A8/A9 Alters the Platelet Proteome in Acute Myocardial Infarction and Is Associated With Changes in Platelet Reactivity.

OBJECTIVE: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment-elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets (R=0.46, P=0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity-an effect abrogated by aspirin. CONCLUSIONS: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

Serotonin-Affecting Antidepressant Use in Relation to Platelet Reactivity.

Depression is an independent risk factor of cardiovascular disease morbidity. Serotonin is a key neurotransmitter in depressive pathology, contained within platelets, and is a weak activator of platelets. Our study assessed the link between platelet reactivity traits, depression, and antidepressant (AD) use in a large population sample. Our study was conducted in the Framingham Heart Study (n = 3,140), and AD use (n = 563) and aspirin use (n = 681) were noted. Depression was measured using the Center for Epidemiological Studies-Depression (CES-D) survey. Platelet reactivity traits were measured across multiple agonists using five distinct assays. We utilized a linear mixed effects model to test associations between platelet traits and depression, adjusting for age, sex, aspirin use, and AD use. Similarly, we analyzed trait associations with any AD use, serotonin-affecting ADs, and norepinephrine-affecting ADs, respectively. There were strong associations with reduced platelet function and AD use, particularly with serotonin-affecting medications. This included lower Optimul epinephrine maximal aggregation (P = 4.87E-13), higher U46619 half maximal effective concentration (P = 9.09E-11), lower light transmission aggregometry (LTA) adenosine diphosphate (ADP) final aggregation (P = 1.03E-05), and higher LTA ADP disaggregation (P = 2.28E-05). We found similar associations with serotonin-affecting ADs in an aspirin-taking subset of our sample. There were no significant associations between platelet traits and depression. In the largest study yet of AD use and platelet function we show that antidepressants, particularly serotonin-affecting ADs, inhibit platelets. We did not find evidence that depressive symptomatology in the absence of medication is associated with altered platelet function. Our results are consistent with AD use leading to platelet serotonin depletions, decreased stability of platelet aggregates, and overall decreased aggregation to multiple agonists, which may be a mechanism by which ADs increase risk of bleeding and decrease risk of thrombosis.

SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity.

Thrombosis of the lung microvasculature 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 that is 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 Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and 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 Niclosamide for COVID-19 therapy.

Traumatic brain injury and hemorrhage in a juvenile rat model of polytrauma leads to immunosuppression and splenic alterations.

BACKGROUND: Traumatic brain injury (TBI) is a common cause of morbidity and mortality. We have previously shown that TBI with a concurrent extra-cranial injury reliably leads to post-injury suppression of the innate immune system, but the impact of this injury on the adaptive immune system is unknown. We present data showing that combined injury reduced immune response as assayed in both blood and spleen samples and that these changes parallel apoptosis in the spleen. To assess the clinical relevance of these changes, we examined lungs for spontaneous bacterial colonization. METHODS: For these studies, prepubescent (28 day old) rats were injured using a controlled cortical impact model and then 25% blood volume removal by arteriotomy, and injured animals were compared with sham injured animals. Blood and spleen samples at post-injury day 1 were incubated with or without immunostimulant and examined for IFN-γ production using an Eli-Spot assay. Spleen samples were also examined for apoptosis using Annexin V staining, and lungs were harvested and plated on blood agar to examine for spontaneous bacterial colonization. RESULTS: Stimulations of whole blood and spleen samples with phorbol 12-myristate 13-acetate/ionomycin (PMA/I) at post-injury day 1 were associated with significant decreases in IFN-γ-positive cells/million in injured animals. Stimulation of whole blood with either PMA/I or pokeweed mitogen led to reduced tumor necrosis factor alpha production. Spleen from injured animals showed a marked increase in apoptosis. Lung samples showed a 300% increase in colonies per plate in injured animals. CONCLUSIONS: These data suggest that the combined injury can lead to adaptive immunosuppression, and our findings further suggest a potential role for the spleen in altering leukocyte function following injury.

Proteome and functional decline as platelets age in the circulation.

BACKGROUND: Platelets circulate in the blood of healthy individuals for approximately 7-10 days regulated by finely balanced processes of production and destruction. As platelets are anucleate we reasoned that their protein composition would change as they age and that this change would be linked to alterations in structure and function. OBJECTIVE: To isolate platelets of different ages from healthy individuals to test the hypothesis that changes in protein content cause alterations in platelet structure and function. METHODS: Platelets were separated according to thiazole orange fluorescence intensity as a surrogate indicator of mRNA content and so a marker of platelet age and then subjected to proteomics, imaging, and functional assays to produce an in-depth analysis of platelet composition and function. RESULTS: Total protein content was 45 ± 5% lower in old platelets compared to young platelets. Predictive proteomic pathway analysis identified associations with 28 biological processes, notably higher hemostasis in young platelets whilst apoptosis and senescence were higher in old platelets. Further studies confirmed platelet ageing was linked to a decrease in cytoskeletal protein and associated capability to spread and adhere, a reduction in mitochondria number, and lower calcium dynamics and granule secretion. CONCLUSIONS: Our findings demonstrate changes in protein content are linked to alterations in function as platelets age. 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.

Platelet inhibition by P2Y12 antagonists is potentiated by adenosine signalling activators.

BACKGROUND AND PURPOSE: P2Y12 receptor antagonists reduce platelet aggregation and the incidence of arterial thrombosis. Adenosine signalling in platelets directly affects cyclic nucleotide tone, which we have shown to have a synergistic relationship with P2Y12 inhibition. Several studies suggest that ticagrelor inhibits erythrocyte uptake of adenosine and that this could also contribute to its antiplatelet effects. We therefore examined the effects on platelet activation of adenosine signalling activators in combination with the P2Y12 receptor antagonists ticagrelor and prasugrel. EXPERIMENTAL APPROACH: Human washed platelets, platelet-rich plasma and whole blood were used to test the interactions between ticagrelor or prasugrel and adenosine or 5'-N-ethylcarboxamidoadenosine (NECA). Platelet reactivity to thrombin, protease-activated receptor 1 (PAR-1) activation or collagen was assessed by a combination of 96-well plate aggregometry, light transmission aggregometry, whole blood aggregometry, ATP release assay and levels of cAMP. KEY RESULTS: The inhibitory effects of ticagrelor and prasugrel on platelet aggregation and ATP release were enhanced in the presence of adenosine or NECA. Isobolographic analysis indicated a powerful synergy between P2Y12 receptor inhibition and adenosine signalling activators. Increased levels of cAMP in platelets were also observed. In all cases, ticagrelor showed similar synergistic effects on platelet inhibition as prasugrel in the presence of adenosine or NECA. CONCLUSION AND IMPLICATIONS: These results indicate that P2Y12 antagonists have a synergistic relationship with adenosine signalling and that their efficacy may depend partly upon the presence of endogenous adenosine. This effect was common for prasugrel and ticagrelor despite reports of differences in their effects upon adenosine reuptake.

Psychosocial impairment following mild blast-induced traumatic brain injury in rats.

Traumatic brain injury (TBI) is associated with increased risk for mental health disorders, impacting post-injury quality of life and societal reintegration. TBI is also associated with deficits in psychosocial processing, defined as the cognitive integration of social and emotional behaviors, however little is known about how these deficits manifest and their contributions to post-TBI mental health. In this pre-clinical investigation using rats, a single mild blast TBI (mbTBI) induced impairment of psychosocial processing in the absence of confounding physical polytrauma, post-injury motor deficits, affective abnormalities, or deficits in non-social behavior. Impairment severity correlated with acute upregulations of a known oxidative stress metabolite, 3-hydroxypropylmercapturic acid (3-HPMA), in urine. Resting state fMRI alterations in the acute post-injury period implicated key brain regions known to regulate psychosocial behavior, including orbitofrontal cortex (OFC), which is congruent with our previous report of elevated acrolein, a marker of neurotrauma and 3-HPMA precursor, in this region following mbTBI. OFC of mbTBI-exposed rats demonstrated elevated mRNA expression of metabotropic glutamate receptors 1 and 5 (mGluR1/5) and injection of mGluR1/5-selective agonist in OFC of uninjured rats approximated mbTBI-induced psychosocial processing impairment, demonstrating a novel role for OFC in this psychosocial behavior. Furthermore, OFC may serve as a hotspot for TBI-induced disruption of psychosocial processing and subsequent mental health disorders.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.