Platelet Activation Research Articles

Association between perioperative platelet distribution width changes and postoperative acute kidney injury in patients with renal insufficiency: a retrospective study

BackgroundAcute kidney injury (AKI) is a major complication following cardiac surgery with a high incidence in those with existing kidney dysfunction. Platelet distribution width (PDW) reflects variability in platelet size and serves as an indicator of platelet activation. Recent investigations linked PDW changes to kidney pathology, suggesting its utility in identifying individuals at risk for AKI, thus necessitating exploration of its predictive value.MethodsPatients with preoperative renal dysfunction [15 ≤ estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m2] who underwent cardiac surgery from January 2018 to December 2021 were retrospectively enrolled. PDW values were measured preoperatively and again upon admission to the ICU immediately after cardiac surgery, with the change in PDW (dPDW) defined as the difference between these two measurements. The primary outcome was postoperative AKI, defined base on the Kidney Disease: Improving Global Outcomes (KDIGO) definition and staging criteria. Multivariate regression models were performed to identify the association between dPDW and AKI and its potential trend. Restricted cubic spline analysis assessed non-linear associations between dPDW and AKI. The Youden index identified an optimal dPDW cut-off for AKI prediction. Subgroup analysis was performed to elucidate the consistency of these associations across the various subgroups.ResultsAKI occurred in 53.10% (513/966) of patients, accompanied by significant PDW increases in cases of AKI (P < 0.001). After adjusting confounders, dPDW was identified as a significant risk factor for AKI [odds ratio (OR) = 1.09, 95% confidence interval (CI): (1.02 ~ 1.16), P = 0.012]. Patients in the highest dPDW quartile (Q4) had a 195% higher AKI risk compared to those in the lowest quartile (Q1) (OR = 2.95, 95% CI:1.78 ∼ 4.90, P < 0.001). Trend analysis indicates that the risk of AKI increased with higher dPDW quartiles (P for trend < 0.001). Youden index showed that dRDW = 1.1 was identified as the optimal diagnostic cut-off value for AKI. Subgroup analyses and interaction tests showed a robust association between dPDW and AKI in all subgroups (P for interaction > 0.05).ConclusionsThis study underscored perioperative PDW changes as a significant predictor of postoperative AKI in patients with renal insufficiency, highlighting its potential in refining risk stratification and management strategies.Clinical trial numberNot applicable for this observational retrospective study.

Platelet antiaggregant activity of Hypericum perforatum extracts

BackgroundIn addition to its potential use as an antidepressant, different studies have shown that Hypericum perforatum (HP) can have antibacterial, antioxidant, and antiviral properties. Despite this potential, most of the therapeutic bioactivities attributed to HP need further scientific support. In a previous study we found an inhibitory effect of HP in platelet activation markers. So, in the present work we investigate the effect of HP in another aspect of platelet function, platelet aggregation. MethodsFirst, the effect of different concentrations of the total alcoholic extract of HP on platelet aggregation was studied in 9 healthy donors by aggregometry methods using collagen as agonist. After that, to obtain dose-effect curves, 3 experiments were carried out independently in a single individual using collagen, adenosine diphosphate and epinephrine as agonists. ResultsOur results reveal a marked inter-individual variability and a significant dose-dependent decrease of aggregation responses induced by collagen in the presence of HP extracts (mean of minimum inhibition: 8% at a dose of 0.068 mg es/ml and mean of maximum inhibition: 43% at a dose of 0.456 mg es/ml). With the results obtained it was possible to obtained a IC50 value of 0.34 mg es/ml. ConclusionsWe can conclude that HP has a dose-dependent inhibitory effect on platelet function (activation and aggregation responses), therefore, our results suggest that HP may be recognized as an herbal medicine with anti-platelet properties.

Enhanced platelet sensitization is accompanied by increased expression of the transporter MRP4 and elevated plasma S1P levels in mild COVID-19 convalescents

Viral infections can lead to platelet activation and hemostatic complications. However, the extent to which platelet reactivity remains altered after convalescence, contributing to long-term health impairments as observed after COVID-19 is not yet fully understood. Therefore, we conducted a cohort study (DRKS00025217) to determine platelet function in individuals convalesced from mild COVID-19. Assays were performed ex vivo with blood from convalescents at 2–15 weeks and 6–10 months after convalescence, focusing on platelet aggregation, activation markers, and thrombin formation. In addition, two other potentially relevant factors for platelet function were examined: the immunomodulatory mediator sphingosine-1-phosphate (S1P) and the platelet expression of the transporter MRP4 (ABCC4). Our findings indicate that robust platelet functions, including platelet aggregation determined by light transmission aggregometry, and thrombin formation, were not altered in convalescents compared to matched control individuals. However, an elevation in subtle platelet activation markers, such as P-selectin surface expression and activation of glycoprotein IIb/IIIa, was observed 2–15 weeks after convalescence. This was accompanied by an increased expression of MRP4 in platelets and significantly elevated levels of S1P in platelet-poor plasma. Our findings suggest increased platelet sensitization and a pro-inflammatory state even after convalescence from mild COVID-19, pointing toward MRP4 and S1P as associated factors.

Novel Calcitonin Gene-Related Peptide (CGRP) Interfering Migraine Therapies and Stroke—A Review

Migraine and stroke are neurological disorders with significant global prevalence and impact. Recent advances in migraine therapy have focused on the calcitonin gene-related peptide (CGRP) pathway. This review examines the shared pathomechanisms between migraine and stroke, with emphasis on the role of CGRP. We analyze the current literature on CGRP’s functions in cerebrovascular regulation, edema formation, neuroinflammation, and neuroprotection. CGRP acts as a potent vasodilator and plays a crucial role in trigeminovascular activation during migraine attacks. In stroke, CGRP has demonstrated neuroprotective effects by improving collateral circulation and reducing ischemia-reperfusion injury. Concerns have been raised about the potential impact of CGRP inhibitors on stroke risk and outcomes. Studies in animals suggest that CGRP receptor antagonists may worsen cerebral ischemia by impairing collateral flow. We discuss the implications of these findings for the use of CGRP-targeting therapies in migraine patients, especially those at increased risk of stroke. Additionally, we explore the complex interplay between CGRP, endothelial function, and platelet activity in both conditions. This review highlights the need for further research to elucidate the long-term cerebrovascular safety of CGRP pathway inhibitors and to identify potential subgroups of migraine patients who may be at higher risk of adverse cerebrovascular events with these novel therapies.

Functional Foods in Preventing Human Blood Platelet Hyperactivity-Mediated Diseases—An Updated Review

Backgrounds/Objectives: Abnormal platelet functions are associated with human morbidity and mortality. Platelets have emerged as critical regulators of numerous physiological and pathological processes beyond their established roles in hemostasis and thrombosis. Maintaining physiological platelet function is essential to hemostasis and preventing platelet-associated diseases such as cardiovascular disease, cancer metastasis, immune disorders, hypertension, diabetes, sickle cell disease, inflammatory bowel disease, sepsis, rheumatoid arthritis, myeloproliferative disease, and Alzheimer’s disease. Platelets become hyperactive in obesity, diabetes, a sedentary lifestyle, hypertension, pollution, and smokers. Platelets, upon activation, can trawl leukocytes and progenitor cells to the vascular sites. Platelets release various proinflammatory, anti-inflammatory, and angiogenic factors and shed microparticles in the circulation, thus promoting pathological reactions. These platelet-released factors also maintain sustained activation, further impacting these disease processes. Although the mechanisms are unknown, multiple stimuli induce platelet hyperreactivity but involve the early pathways of platelet activation. The exact mechanisms of how hyperactive platelets contribute to these diseases are still unclear, and antiplatelet strategies are inevitable for preventing these diseases. Reducing platelet function during the early stages could significantly impact these diseases. However, while this is potentially a worthwhile intervention, using antiplatelet drugs to limit platelet function in apparently healthy individuals without cardiovascular disease is not recommended due to the increased risk of internal bleeding, resistance, and other side effects. The challenge for therapeutic intervention in these diseases is identifying factors that preferentially block specific targets involved in platelets’ complex contribution to these diseases while leaving their hemostatic function at least partially intact. Since antiplatelet drugs such as aspirin are not recommended as primary preventives, it is essential to use alternative safe platelet inhibitors without side effects. Methods: A systematic search of the PUBMED database from 2000 to 2023 was conducted using the selected keywords: “functional foods”, “polyphenols”, “fatty acids”, “herbs”, fruits and vegetables”, “cardioprotective agents”, “plant”, “platelet aggregation”, “platelet activation”, “clinical and non-clinical trial”, “randomized”, and “controlled”. Results: Potent natural antiplatelet factors have been described, including omega-3 fatty acids, polyphenols, and other phytochemicals. Antiplatelet bioactive compounds in food that can prevent platelet hyperactivity and thus may prevent several platelet-mediated diseases, including cardiovascular disease. Conclusions: This narrative review describes the work during 2000–2023 in developing functional foods from natural sources with antiplatelet effects.

Hyperexpression of PTAFR and PF4 as Possible Platelet Risk Biomarkers in Patients With COVID-19.

SARS-CoV-2 infection presents different severity levels that suggest the influence of genetic factors on the clinical outcome of the disease. In cases of severe COVID-19, the presence of elevated coagulation markers, increased platelet activation and aggregation and the risk of thrombotic complications are described. Given the participation of these cells in several serious viral infections and their negative role when associated with a prothrombotic response, it is important to understand the mechanistic role of SARS-CoV-2 in platelet physiology. This study evaluated the hyperexpression of platelet-activating factor receptor (PTAFR) and platelet factor 4 (PF4) in unvaccinated and hospitalized patients with COVID-19. The study included 43 COVID-19 patients stratified according to WHO guidelines. Subsequently, the expression of the PTAFR and PF4 genes were evaluated using the real-time quantitative PCR and their possible correlation with the severity of the disease and clinical variables including hospitalization, outcome, sex, age and laboratory parameters (platelet count, INR and D-dimer). The analysis demonstrated a significant (p<0.05) hyperexpression of these genes COVID-19 patients (n=43) compared to healthy controls. Expression of these genes in patients was not statistically significant (p>0.05) different between patients stratified according to clinical variables. The expression of PTAFR and PF4 suggests an important molecular pathway in the pathophysiology of the disease and may be valuable platelet biomarkers to indicate increased risk in patients with COVID-19 who require hospital care, contributing to personalized intervention strategies and improving their clinical management.

CS585, a novel prostacyclin receptor agonist, demonstrates sustained efficacy in vivo in the prevention of thrombosis

Abstract Background Uncontrolled platelet activity resulting from cardiovascular disease can lead to occlusive thrombi, culminating in myocardial infarction or stroke. Despite major advances in anti-platelet therapeutics, many patients remain at risk. Although prostacyclin (IP) receptor agonists are currently used for the treatment of pulmonary arterial hypertension (PAH), activation of the IP receptor has been shown to decrease platelet reactivity. However, engaging the IP receptor as a therapeutic target was previously considered unviable due to the lack of a sustained effect of current IP agonists in the blood. The development of an IP agonist with sustained effects in the blood would represent a novel prevention strategy in targeting platelet hyperreactivity and thrombosis. Purpose Assess the stability profile of CS585, an IV and orally bioavailable IP agonist, compared to FDA-approved IP agonists iloprost and selexipag, with regards to sustained activity in vivo in the blood in limiting platelet activation and thrombosis. Methods Using ex vivo and in vivo mouse models, we assessed the timeframe of effect of CS585, iloprost and selexipag in blood. Blood was drawn from wild-type mice 24 hours following a single IV dose of CS585, iloprost or selexipag. Platelet adhesion and blood clotting were measured using perfusion flow chamber at arterial shear and total thrombus activation system (T-TAS), respectively. In vivo, platelets and fibrin were labelled 24 hours post IV or oral administration of CS585, iloprost or selexipag, and accumulation at the site of injury was measured using the cremaster arteriole injury thrombosis assay. Results Blood drawn from mice administered CS585 was observed to show a decrease in platelet adhesion and clot formation in the perfusion flow chamber and T-TAS assays. The effects of iloprost and selexipag are no longer observed at 24 hours post-administration. Administration of CS585 resulted in sustained inhibition of platelet accumulation and fibrin formation in the laser-induced cremaster arteriole thrombosis assay up to 18 hours post-administration. In mice administered iloprost, a decrease in thrombus formation was observed 10 minutes post-administration, but thrombus size returned to vehicle levels by 4 hours. Selexipag-dosed mice demonstrated inhibition of thrombus formation up to 4 hours, but effects were no longer observed at 18 hours. Conclusions CS585, a novel IP agonist, inhibits platelet activation and clot formation up to 24 hours post-administration. In both in vivo and ex vivo models, we demonstrate the sustained effects of CS585. In contrast, current FDA-approved IP agonists do not possess an effect profile sufficient to target the IP receptor in blood. By overcoming the sustainability challenge facing IP agonists, CS585 represents a novel approach to anti-platelet treatment of thrombotic diseases, which could include thrombosis, VTE, secondary prevention after MI and stroke, and PAH.

Direct IL-6 inhibition prevents arterial thrombosis by reducing collagen-mediated platelets activation

Abstract Introduction Interleukin 6 (IL-6) has a pivotal role in atherothrombosis. Yet, targeting IL-6 to prevent atherothrombotic events still requires validation of efficacy and safety. The recent phase 2 RESCUE trial reported that direct inhibition of IL-6 ligand by the monoclonal antibody (mAb) Ziltivekimab is safe in patients with elevated cardiovascular risk. Purpose This project investigated the effects of direct IL-6 inhibition on arterial thrombosis, and assessed the underlying cellular mechanisms. Methods Three month old C56Bl/6 male mice received very-low dose LPS i.p. for 4 weeks. During the last week they were randomized to receive in addition either anti-mouse IL-6 mAb 200 μg i.p. every other day or IgG1 isotype control. Then thrombosis of left common carotid artery (LCCA) was induced by laser injury under anesthesia. Platelets of treated mice were isolated and stimulated with either adenosine di-phosphate (ADP), collagen-related peptide (CRP) or thrombin receptor activating peptide (TRAP). Platelets activation was measured by flow-cytometry, as expression of P-selectin and JON/A.The effect of direct IL-6 inhibition was confirmed in patients with stable coronary artery disease (sCAD) by ex-vivo incubation of isolated platelets with either anti-human IL-6 mAb 1.5 μg/mL or IgG1 isotype control. Platelet activation was measured by flow-cytometry, as expression of P-selectin. Results Mice treated with anti-IL6 antibody displayed a significantly longer time-to-occlusion after the LCCA (Figure 1), without any significant difference in coagulation parameters. After stimulation with CRP, platelets were significantly activated in control mice, but not in mice treated with anti-IL6 mAb. Similarly, activation of isolated platelets from patients with sCAD was significantly reduced (Figure 2) by the ex-vivo treatment with anti-IL6 mAb. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition. Conclusion The direct inhibition of IL-6 reduces platelets reactivity to collagen, thereby blunting arterial thrombosis upon endothelial damage. These results provide a potential mechanism to explain the reduction of cardiovascular risk associated to direct IL-6 inhibition.Figure 1Figure 2

The implantation of a left ventricular assist device causes changes in the aortic miRNome and proteome - an integrative analysis

Abstract Background Left ventricular assist devices (LVAD) have been widely used and accepted to treat patients with end-stage heart failure. LVAD non-physiological blood flow velocity, wall shear stress distribution, vorticity current intensity, and vorticity flow generation affect the pathophysiology of vascular changes in aortic tissue. Purpose In this study, we performed multi-omics-based analysis of the aorta to identify molecular markers that could clarify vascular remodeling during LVAD support. Methods Aortic specimens from 48 patients (pair matched samples N=96) were excised during implantation and explantation of LVAD. Small RNA-Seq screening using Next Generation Sequencing and proteomic profile using Data-Independent Acquisition mass spectroscopy were conducted. Experimental and computational approaches, proteomics and transcriptomics data, were integrated and bioinformatics analyses were performed. Results The principal component analysis of miRNome and permutational multivariate analysis of variance of proteomic profile clearly segregated samples before and after LVAD support. We identified a total of 113 differently expressed (DE) miRNAs (61 up-regulated; Padj&amp;lt;0.05 and log2 fold change&amp;gt;|0.5|) after long-term LVAD support. Proteomic analysis identified in total 93 protein significantly changed (47 increased; Padj&amp;lt;0.05 and log2 fold change&amp;gt;|0.5|). Correlation analysis identified strong association between 270 proteins and 66 DE-miRNAs (Padj&amp;lt;0.05 and correlation coefficient&amp;gt;|0.7|). Gene Set Enrichment Analysis revealed, that predicted target genes of DE-miRNAs were mainly engaged in axon guidance, regulation of actin cytoskeleton, endocytosis, and MAP signaling pathway (Padj&amp;lt;0.05). DE-proteins participated mainly in ECM-receptor interaction, focal adhesion, and platelet activation (Padj&amp;lt;0.05). Conclusion Our study presents a network-based method of integrating microRNAs and proteome data and reveals molecular signatures that reflects aortic vascular remodeling due to LVAD treatment.PCA of miRNA DESeq2PCA of protemic profile

Neutrophil-Mimetic Upconversion Photosynthetic Nanosystem Derived from Microalgae for Targeted Treatment of Thromboembolic Stroke.

Thromboembolic stroke constitutes the majority of brain strokes, resulting in elevated mortality and morbidity rates, as well as significant societal and economic burdens. Although intravenous thrombolysis serves as the standard clinical treatment, its narrow therapeutic window and the inflammatory response induced by tissue plasminogen activator (tPA) administration limit its efficacy. In the initial stages of stroke, the abrupt cessation of blood flow leads to an energy metabolism disorder, marked by a substantial decrease in adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) levels, causing irreversible damage to neural cells. In this study, we introduce a neutrophil-mimetic, microalgae-derived upconversion photosynthetic nanosystem designed for targeted treatment of thromboembolic stroke. This system features upconversion nanoparticles coated with a thylakoid membrane and wrapped in an activated neutrophil membrane, further decorated with ROS-responsive thrombolytic tPA on its surface. The neutrophil-mimetic design facilitates high targeting specificity and accumulation at the thrombus site after intravenous administration. Upon exposure to elevated levels of reactive oxygen species (ROS) at the thrombus location, the nanosystem promptly demonstrated potent thrombolytic efficacy through the surface-modified tPA. Furthermore, near-infrared II (NIR-II) laser irradiation activated the generation of ATP and NADPH, which inhibited inflammatory cell infiltration, platelet activation, oxidative stress, and neuronal injury. This constructed nanoplatform not only showcases exceptional targeting efficiency at the stroke site and controllable release of the thrombolytic agent but also facilitates ATP/NADPH-mediated thrombolytic, anti-inflammatory, antioxidative stress, and neuroprotective effects. Additionally, it offers valuable insights into the potential therapeutic applications of microalgae-based derivatives in managing thromboembolic stroke.

Effects of rivaroxaban on vascular endothelial function and circulating endothelial progenitor cells: a randomized controlled trial

Abstract Background Combination of low dose rivaroxaban with aspirin was shown to reduce major adverse cardiovascular events when compared to aspirin monotherapy in COMPASS trial. The beneficial effect of rivaroxaban beyond factor Xa inhibition remains unknown. Purpose This randomized controlled trial explored the effects of rivaroxaban on vascular endothelial function and circulating endothelial progenitor cells in patients with stable atherosclerotic disease. Methods An investigator-initiated, investigator-blinded, single-center randomized clinical trial was conducted from May 2021 to August 2023. 142 patients were randomized in 1:1 ratio to receive rivaroxaban 2.5mg twice daily plus aspirin 100mg once daily (Treatment Group) or aspirin 100mg once daily (Control Group) and had follow-up for 3 months. The primary outcome was change of flow mediated dilation (FMD) from baseline to 3-month follow-up. Key secondary outcomes included change of circulating endothelial progenitor cells (CD45+/34+/133+, CD45+/34+/KDR+), platelet activation marker (CD41+/62p+), inflammatory marker (CD14+/11b+) and thrombo-inflammatory maker (CD14+/42b+) measured by flow cytometry, as well as platelet function (aspirin reaction unit, ARU) measured by blood biochemistry. Intention-to-treat analysis was performed with analysis of covariance (ANCOVA) and P &amp;lt; 0.05 was considered statistically significant. Results Overall, 142 patients were enrolled in the study. One patient was excluded from analysis due to protocol deviation. Among the remaining 141 patients (78.8% male and mean age 69±9 years), there were 71 in Treatment Group and 70 in Control Group. The recruited patients had a high prevalence of cardiovascular comorbidities including 76.6% with hypertension, 50.4% with hyperlipidemia, 45.4% with diabetes, and 92.2% with percutaneous coronary intervention. At 3 months, Treatment Group had greater FMD improvements (+2.18±5.66% vs +1.57±5.07%, P=0.03), and more reduction in platelet activation marker (-0.33±13.55% vs +5.78±21.60%, P=0.01). There was no significant change in circulating endothelial progenitor cells, inflammatory marker, thrombo-inflammatory maker, and platelet function. Minor bleeding occurred in 2 patients (2.82%) in Treatment Group and none in Control Group (P&amp;gt;0.05). Conclusion In patients with stable atherosclerotic disease, addition of rivaroxaban to aspirin was well tolerated, and it improved vascular endothelial function and reduced platelet activation.

Dual pathway inhibition in patients with atherosclerosis with and without heart failure: insights from the XATOA Registry

Abstract Introduction Patients with atherosclerotic cardiovascular disease (ASCVD) are at a high-risk of experiencing a major adverse cardiovascular event, with 1 in 10 individuals experiencing an event within four years (1). To address this risk of secondary cardiovascular events, there have been renewed efforts to enhance secondary prevention therapies through the application of guideline-directed medical therapy. One secondary prevention strategy is the use of dual pathway inhibition (DPI) therapy with aspirin and vascular-dose rivaroxaban (2.5mg twice daily), which simultaneously targets both the platelet activation and thrombin generation pathways. Patients with concomitant ASCVD and heart failure (HF) are a subgroup with a higher risk for ischemic events, but have not been adequately represented in recent clinical trials. Purpose To explore the risks and benefits of DPI therapy in a generalizable population of patients with concomitant ASCVD and HF. Methods The Xarelto plus Acetylsalicylic acid Treatment patterns and Outcomes in patients with Atherosclerosis (XATOA) registry is a prospective, multicentre registry of patients with either coronary artery or peripheral artery disease that were initiated on DPI (2). The primary endpoint was a composite of cardiovascular death, myocardial infarction or stroke and the safety outcome was major bleeding. Multivariable logistic regression was performed to assess the association of HF status and ejection fraction (EF) on the outcomes of interest. Results Of 5532 participants, 4022 (72.7%) had documentation of HF status. Of those 873 (21.5%) had a history of heart failure (EF &amp;gt;40% - 461; EF ≤40% - 181, EF unknown – 231). Over a median follow-up of 465 days (IQR – 372-576), the primary outcome occurred in 4.9% of participants with HF compared to 2.4% in those without HF (aHR 1.57 95% CI 1.02-2.41). The safety outcome was similar in patients with and without HF (0.9% versus 1.11%; aHR 0.7 95% CI 0.31-1.67). Among patients with HF, those with an EF of ≤40% demonstrated a non-significant trend towards higher rates of adverse events including MACE (8.8% versus 3.5%; aHR 1.38; 95% CI 0.59-3.22) compared to those with an EF of &amp;gt; 40%. Conclusion In a generalizable cohort of patients with atherosclerotic disease and HF, the use of DPI is associated with similar outcomes to those observed in recent randomised controlled clinical trials.Table 1 - Demographics by HF statusFigure 1 - MACE by HF status

Postpartum cardiac reverse remodeling is reflected in urine proteome

Abstract Background The impact of postpartum cardiac reverse remodeling (RR) on urinary proteome, particularly in pregnant women with cardiovascular risk (CVR) factors who show long-term increased risk of cardiovascular disease and mortality is unknown. Purpose To profile the urinary proteome in pregnant women with/without CVR factors to identify proteins associated with postpartum RR. Methods Our study included a prospective cohort of 32 healthy and 27 obese and/or hypertensive and/or diabetic pregnant women who underwent transthoracic echocardiography, pulse-wave-velocity, and urine collection at the 3rd trimester and 6-months postpartum. Shotgun HPLC-MS/MS profiled proteins. Generalized linear mixed-effects models were used to identify associations between urinary proteins and left ventricle mass (LVM), a surrogate of RR. Results The presence of at least one CVR factor was associated with worse LVM RR. We identify 6 (alpha-galactosidase A [GLA], sialate O-acetylesterase [SIAE], trans-golgi network integral membrane protein 2 [TGOLN2], sortilin [SORT1], desmoglein-2 [DSG2] and neuroblast differentiation-associated protein [AHNAK]) and 11 (serotransferrin [TF], inter-α-trypsin inhibitor heavy chain 4 [ITIH4], complement C3 [C3], transketolase [TKT], fibronectin [FN1], ubiquitin-like modifier-activating enzyme 1 [UBA1], cell division control protein 42 homolog [CDC42], glutathione s-transferase P1 [GSTP1], sodium/potassium-transporting ATPase subunit alpha-1 [ATP1A1], ADP-ribosylation factor 1 [ARF1] and pro-cathepsin H [CTSH]) proteins associated with high and low LVM regression, respectively. These proteins were functionally linked with insulin-like growth factor (IGF) transport and uptake regulation by IGF binding-proteins, platelet activation, signaling and aggregation and the immune system’s activity. The concentration of IGF-1 in urine samples was associated with low LVM regression after delivery. Conclusions Urine proteome reflects postpartum cardiac RR. Specifically, eight urine proteins (GSTP1, ARF1, FN1, SORT1, SIAE, GLA, ITIH4 and DGS2) show clinical value in identifying women at higher risk for incomplete postpartum RR. In addition, the association between IGF and postpartum LVM regression was validated.

Single-cell mass cytometry analysis unveils prothrombotic expression and heterogeneity in essential thrombocythemia

Abstract Background/Introduction Essential thrombocythemia (ET) is recognized for its hallmark elevated platelet count and a consequent increased risk of thromboembolic events including coronary events. Despite its clinical significance, the pathophysiological mechanisms predisposing ET patients to thrombosis remain largely undefined. Purpose The aim of this pioneering study was to shed light on the increased thrombotic risk in ET by conducting a detailed phenotypical characterization of platelet expression using single-cell mass cytometry, comparing ET patients to age- and sex-matched healthy controls. Methods Platelet samples from six patients diagnosed with ET and six healthy individuals were analyzed. This analysis utilized an extensive panel of 18 transmembrane regulators pivotal for platelet function and activation. Measurements were taken both at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). Advanced clustering analysis via the FlowSOM algorithm helped identify specific subclusters of platelets with a prothrombotic expression profile. Results Our investigation revealed a significant overexpression of the activation marker CD62P (P-Selectin, p=0.049) and of the collagen receptor GPVI (p=0.044) in non-stimulated ET platelets (Figure 1A-C). Conversely, a notable decrease in expression was observed for the fibrinogen receptor GPIIb/IIIa units CD41 (p=0.036) and CD61 (p=0.044), as well as for the von Willebrand factor receptor CD42b (p=0.044). The FlowSOM analysis distinguished two prothrombotic subclusters of ET platelets, particularly highlighting one cluster, which was significantly overrepresented in ET (22.13% of total ET platelets versus 2.94% in controls, p=0.035, Figure 1D-E). Conclusion This study presents compelling evidence of a distinct prothrombotic phenotype in ET, marked by significant alterations in platelet expression profiles. The overexpression of CD62P and GPVI, alongside the underexpression of CD41, CD61, and CD42b, underscores the complex pathophysiological landscape of ET. Importantly, we identified a specific subcluster, particularly overrepresented in ET, with a specific prothrombic signature. Our findings, while only hypothesis generating, propose GPVI as a potential therapeutic target to mitigate thrombosis in ET patients, paving the way for targeted interventions in this high-risk population.

Sex differences in RNA transcripts in dilated cardiomyopathy

Abstract Introduction Dilated cardiomyopathy (DCM) is the most common cardiomyopathy and a major cause of heart failure. Differences in clinical presentation and phenotype exist in men and women but the reason for this is not clear. The analysis of the RNA transcripts (mRNA sequencing) offers an opportunity to provide a link between the genome, the proteome, and the phenotype. The aim of this study was to explore sex differences using mRNA sequencing in patients with DCM. Method Our database, an observational prospective cohort study, includes patients referred for suspected cardiomyopathy. The database includes data on patients’ characteristics, symptoms, diagnostic procedures, treatments, cardiac devices, and outcome. Endomyocardial biopsy from the right ventricle was collected and total RNA, including miRNA and other small RNA, was isolated. RNA sequencing and library preparation was performed. Following quality check, reads were aligned to GRCh38 reference genome by STAR (v2.7.10a). Sex-specific differential expression analysis between individuals diagnosed with DCM (cases) versus non-confirmed cardiomyopathy (controls) was performed using DESeq2 (v.1.38.3) adjusting for age, RNA integrity number and systolic blood pressure. After controlling for multiple testing using the Benjamini-Hochberg method, genes with a corrected P-value &amp;lt; 0.05 were considered as being differentially expressed. Gene-set enrichment analysis on differentially expressed genes was performed via Enrichr tool using Descartes cell types and tissues, KEGG biological pathways, DisGeNET, and Gene Ontology Biological Processes (GOBP). Results Patients´characteristics in women and men with DCM is presented in table 1. There was no difference in outcome in men and women (data not shown). Differential expression analyses with transcriptomic data comparing DCM (n=43) versus controls (n=22) stratified by sex (DCM n= 9, (20.9%) women and controls n=7, (30.4%) women) was performed. There was no overlap between differentially expressed genes between sexes (Figure 1). In men gene set enrichment analyses showed that differentially expressed genes were related to pathways involved in dilated and hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy (KEGG). Interestingly, these analyses revealed an enrichment of genes related to platelet biology, namely precursors (Descartes), activation (GOBP, KEGG) and platelet disorder (DisGeNET). In women, HLA-DQB1 was more expressed in DCM. HLA-DQB1 is associated with celiac disease, narcolepsy, and type 1 diabetes. Consistently, the enrichment analyses showed an enrichment of genes involved in autoimmune diseases (DisGeNET). Conclusion Women and men with DCM exhibit differently activated RNA transcripts although no difference in phenotype was observed. This may indicate that although DCM can be caused by similar factors in women and men the transcriptome is differently activated which warrants further investigation.Table 1.

Elderly patients are hyperresponsive to potent P2Y12 inhibitors

Abstract Background Aging has recently been associated with increased basal platelet activation and adenosine diphosphate (ADP) hyperreactivity on the one hand, but decreased platelet response to thrombin receptor stimulation on the other hand in individuals without antiplatelet therapy. In the current study, we investigated platelet response to agonist stimulation in elderly patients (70 years or older) on dual antiplatelet therapy with potent P2Y12 inhibitors. Methods Platelet aggregation in response to arachidonic acid (AA), ADP, collagen, the protease-activated receptor (PAR)-1 agonist SFLLRN and the PAR-4 agonist AYPGKF was assessed by multiple electrode aggregometry in 79 prasugrel- and 77 ticagrelor-treated patients 3 days after acute percutaneous coronary intervention. Results In the overall study population (n=156), patients aged ≥70 years (n=33) had lower platelet aggregation in response to AA, ADP and SFLLRN than younger patients (all p&amp;lt;0.05). In prasugrel-treated patients (n=79), those aged ≥70 years (n=13) showed significantly lower platelet aggregation in response to all agonists compared to younger patients (all p&amp;lt;0.05). In contrast, in ticagrelor-treated patients (n=77), those aged ≥70 years (n=20) only had significantly lower ADP-inducible platelet aggregation than younger patients (p=0.03), whereas platelet aggregation in response to AA, collagen, SFLLRN and AYPGKF was similar between elderly and younger patients on ticagrelor (all p&amp;gt;0.05). Among patients aged ≥70 years, prasugrel-treated patients showed significantly lower platelet aggregation in response to AA, collagen and AYPGKF than those receiving ticagrelor (all p&amp;lt;0.05). Conclusion Patients aged ≥70 years on potent P2Y12 inhibitors exhibit increased inhibition of ADP-inducible platelet aggregation. In addition, elderly patients on prasugrel show a significantly lower response to AA, collagen, SFLLRN and AYPGKF than younger patients.Platelet aggregation prasugrel patientsPlatelet aggregation ticagrelor patients

Platelet-large cell ratio predicts no-reflow in patients undergoing primary coronary angioplasty for ST-segment elevation myocardial infarction

Abstract Background Increased platelet indices have been shown to reflect higher degree of platelet activation and have been related to poorer short- and long-term outcomes in patients presenting with ST-segment elevation myocardial infarction (STEMI). Purpose We aimed to evaluate the ability of a validated marker of platelet activation (i.e., the platelet-large cell ratio [P-LCR]) to predict the occurrence of no-reflow in patients undergoing primary percutaneous coronary intervention (pPCI) for STEMI. The ability of P-LCR to predict other markers of thrombotic charge (i.e., use of glycoprotein IIb/IIIa inhibitors and/or thrombus aspiration) was also investigated. Methods Use of glycoprotein IIb/IIIa inhibitors and/or thrombus aspiration, occurrence of the ‘no-reflow’ phenomenon and P-LCR values on admission were recorded in 836 consecutive patients treated by pPCI for STEMI. Patients’ demographic data and symptom onset-to-cardiac catheterization laboratory time intervals were also recorded. Correlations between P-LCR values and the presence of thrombotic charge markers were evaluated. Results No-reflow occurred in 124 (14.8%) of the study patients; glycoprotein IIb/IIIa inhibitors and thrombus aspiration were used in 228 (27.3%) and in 289 (34.5%) of patients, respectively. Mean P-LCR values were 27.1 ± 7.6%. P-LCR values on admission were significantly higher in patients who presented no-reflow than in those who did not (29.1 ± 5.6% vs. 25.3 ± 5.8%, p = 0.03). Similarly, P-LCR were significantly higher in patients in whom glycoprotein IIb/IIIa inhibitors (29.5 ± 6.1% vs. 25.2 ± 5.3%, p = 0.04) and/or thrombus aspiration (28.4 ± 5.7% vs. 24.8 ± 5.4%, p = 0.03) were used. A P-LCR value ≥26.9% predicted the occurrence of no-reflow with 70.9% sensitivity and 55.4% specificity (p = 0.04). Conclusions In the present cohort, P-LCR values on admission predicted the presence of thrombotic charge markers in patients undergoing pPCI for STEMI. Evaluating P-LCR on admission could help identify patients at increased risk of no-reflow, in whom more intensive antiplatelet strategies may be of interest.

Pullulan/Collagen Scaffolds Promote Chronic Wound Healing via Mesenchymal Stem Cells

This study investigated the development of Pullulan/Collagen nanofiber scaffolds integrated with mesenchymal stem cells (MSCs) to enhance chronic wound healing. The combination of these biopolymers aims to optimize the scaffold properties for cell growth, viability, and tissue regeneration. Materials and Methods: Pullulan, Collagen, and Pullulan/Collagen composite nanofibers were fabricated using electrospinning. The fibers were characterized using scanning electron microscopy (SEM) to determine the fiber diameter, and Fourier-transform infrared spectroscopy (FTIR) was employed to assess the molecular interactions. Cell viability was evaluated using MSCs cultured on the scaffolds and apoptosis assays were conducted to assess cell health. Distilled water was used as the solvent to maximize biocompatibility. Results: SEM analysis revealed that Pullulan nanofibers exhibited a larger average diameter (274 ± 20 nm) compared to Collagen fibers (167.03 ± 40.04 nm), while the Pullulan/Collagen composite fibers averaged 280 ± 102 nm. FTIR confirmed the molecular interactions between Pullulan and Collagen. Regarding biocompatibility, the Pullulan/Collagen scaffold demonstrated superior cell viability at 99% compared to 91% for Pullulan alone. Apoptosis assays indicated significantly lower necrosis rates for the composite scaffold (1.29%) than for the Pullulan-only scaffolds (2.35%). Conclusion: The use of distilled water as a solvent played a critical role in increasing cell viability and facilitating healthy proliferation of MSCs without cellular damage. Additionally, the reduced platelet activation and macrophage activity (0.75-fold for both) further supported the biocompatibility of the Pullulan/Collagen scaffold, demonstrating its potential for tissue engineering and chronic wound healing applications.

From bench to bedside and back to bench: investigating the role of platelet heterogeneity in coronary artery disease

Abstract Background/Introduction Platelets exhibit considerable heterogeneity in RNA content, size, and thrombogenicity. Our preliminary investigation of the transcriptome of the RNA-rich reticulated platelets(RPs) in healthy donors revealed an enrichment of prothrombotic transcripts compared to mature platelets(MPs). Recently, we validated the prognostic role of elevated RPs at clinical level in a modern cohort of ACS patients treated with potent P2Y12 inhibitors. However, the pathophysiology of RP hyperreactivity remains unclear, particularly its correlation with adverse events and cardiovascular death. Purpose To study the biology of RPs in patients with chronic coronary syndrome(CCS) and to elucidate their role in disease progression. Methods RPs were isolated from peripheral blood of CCS patients(N=20) and compared with MPs. Cell viability and reactivity of RPs were quantified by FACS. Deep transcriptomic profiling was performed using post-sorting bulk RNA sequencing of RPs and MPs and analyzed with ad-hoc bioinformatic pipelines. Proteomic profiling using mass cytometry(CyTOF) was used to validate the transcriptomic findings with single cell resolution. Results FACS analysis confirmed RPs hyperreactivity showing increased P-Selectin expression upon activation with TRAP, ADP and Collagen(Fig. 1A). Total RNA-sequencing detected 2213 differentially regulated genes with an enrichment in RPs of key functional transcript such as, the von Willebrand Factor VWF(p=3.06*10-33), the thromboxane receptor TBXA2R(p=2.07*10-29) and the collagen receptor GP6(p=1.29*10-38, Fig. 1B-C). Gene ontology and gene set enrichment analyses detected an upregulation of relevant categories as "Regulation of platelet activation" and "Platelet aggregation"(Fig. 1D-F). In addition, we detected a novel alternative splicing event on the collagen receptor transcript GP6 upregulated in RPs(Fig. 1G). We detected 33 differentially regulated miRNAs, including miR-409, miR-134 and miR-432, which have been already linked to prothrombotic phenotypes(Fig. 1H). Additionally, we detected an enrichment of circular RNAs in RPs compared to MPs with several circular RNA upregulated in RPs, that have not been described before(Fig. 1I-K). Mass cytometry detected a significant upregulation in RPs of relevant proteins including the collagen receptor GPVI(p=8.98*10-12, Fig.2) and the thrombin receptor PAR1(p=5.21*10-11). Validation assays in an independent cohort detected an upregulation of the PI3K/AKT pathway in RPs which is downstream of the receptor PAR1 and GP6. Conclusion This study represents the first comprehensive multiomic characterization of RPs in CCS patients, providing insights into their prothrombotic phenotype and into their correlation with cardiovascular events. These findings suggest potential avenues for tailored therapeutic interventions targeting the identified upregulated pathways in patients with elevated RPs, warranting further investigation at the clinical level.

Platelet Glycoprotein Ibα Cytoplasmic Tail Exacerbates Thrombosis During Bacterial Sepsis

Septic patients, coupling severe disseminated intravascular coagulation (DIC) and thrombocytopenia, have poor prognoses and higher mortality. The platelet glycoprotein Ibα (GPIbα) is involved in thrombosis, hemostasis, and inflammation response. However, it remains unclear whether the GPIbα cytoplasmic tail regulates sepsis-mediated platelet activation and inflammation, especially in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections. Using a mouse model of S. aureus-induced bacteremia, we found that both 10 amino acids of GPIbα C-terminal sequence deficiency and pharmacologic inhibition of protein kinase C (PKC) alleviated pathogenesis by diminishing platelet activation and aggregate formation. Furthermore, the GPIbα cytoplasmic tail promoted the phagocytosis of platelets by Kupffer cells in vivo. The genetically deficient GPIbα cytoplasmic tail also downregulated inflammatory cytokines and reduced liver damage, ultimately improving the survival rate of the septic mice. Our results illustrate that the platelet GPIbα cytoplasmic domain exacerbates excessive platelet activation and inflammation associated with sepsis through a PKC-dependent pathway. Thus, our findings provide insights for the development of effective therapeutic strategies using PKC inhibitor treatment against bacterial infection.