Platelet mass cytometry reveals dysregulation of prothrombotic pathways in essential thrombocythemia.

Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.

Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.

Be prepared for the unexpected: Mind-stretching in ST-elevation myocardial infarction with situs inversus totalis.

Situs inversus totalis is a rare diagnosis, but the likelihood of experiencing myocardial infarction is presumed to be comparable to the general population average. In individuals exhibiting situs inversus with suspected myocardial infarction, ECG recording, including right precordial leads, is crucial for diagnostic assessment. Coronary angiography and intervention should be performed with standard equipment using inverted maneuvers and radiographic projections.

Right Heart Remodeling and Outcomes in Patients With Tricuspid Regurgitation: A Literature Review and Meta-Analysis.

BACKGROUND: Functional tricuspid regurgitation (TR) can develop either because of right ventricular (RV) remodeling (ventricular functional TR) and/or right atrial dilation (atrial functional TR). OBJECTIVES: This meta-analysis aimed to investigate the association between right heart remodeling and long-term (>1 year) all-cause mortality in patients with significant TR (at least moderate, ≥2+). METHODS: MEDLINE, ISI Web of Science, and SCOPUS databases were searched. Studies reporting data on at least 1 RV functional parameter and long-term all-cause mortality in patients with significant TR were included. This study was designed according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) requirements. RESULTS: Out of 8,902 studies, a total of 14 were included, enrolling 4,394 subjects. The duration of follow-up across the studies varied, ranging from a minimum of 15.5 months to a maximum of 73.2 months. Overall, long-term all-cause mortality was 31% (95% CI: 20%-41%; P ≤ 0.001). By means of meta-regression analyses, an inverse relation was found between tricuspid annular plane systolic excursion (11 studies enrolling 3,551 subjects, -6.3% [95% CI: -11.1% to -1.4%]; P = 0.011), RV fractional area change (9 studies, 2,975 subjects, -4.4% [95% CI: -5.9% to -2.9%]; P < 0.001), tricuspid annular dimension (7 studies, 2,986 subjects, -4.1% [95% CI: -7.6% to -0.5%]; P = 0.026), right atrial area (6 studies, 1,920 subjects, -1.9% [95% CI: -2.5% to -1.3%]; P < 0.001) and mortality. CONCLUSIONS: RV dysfunction parameters are associated to worse clinical outcomes in patients with TR, whereas right atrial dilatation is linked to a better prognostic outcome. Further studies are needed to unravel the pathophysiological differences within the functional TR spectrum. (Right heart remodeling and outcomes in patients with tricuspid regurgitation; CRD42023418667).

Reticulated Platelets Predict Cardiovascular Death and Adverse Events in Coronary Artery Disease: A Systematic Review and Meta-analysis.

BACKGROUND: The pro-thrombotic immature or reticulated platelets (RPs) are known to be elevated in high-risk patients and in different pathological settings. It has been shown that RPs correlate with an insufficient antiplatelet response to antiplatelet agents. RPs are emerging novel predictors of adverse cardiovascular events in cardiovascular disease. This study, using the totality of existing evidence, evaluated the prognostic role of RPs in patients with coronary artery disease. METHODS: We performed a systematic review and meta-analysis including trials of acute and chronic coronary syndrome reporting clinical outcomes according to RPs levels in the peripheral blood. We compared patients with elevated RPs (RPshigh) to patients without elevated RPs (RPslow). Odds ratios (ORs) and 95% CIs were used as metric of choice for treatment effects with random-effects models. The primary endpoint was major adverse cardiovascular and cerebrovascular events (MACCE). Secondary endpoints were cardiovascular death, myocardial infarction, ischemic stroke, urgent coronary revascularization and bleedings. RESULTS: A total of 7 studies, including 2213 patients, were included. The risk for MACCE was significantly higher in RPshigh compared to RPslow patients (OR 2.67 [1.87; 3.81], I2 = 43.8%). RPshigh were associated with cardiovascular death (OR 2.09 [1.36; 3.22], I2 = 40.4%). No associations for RPshigh were detected with the other singular components of MACCE: myocardial infarction (OR 1.73 [0.89; 3.38] I2 = 60.5%) and stroke (OR 1.72 [0.59; 4.96] I2 = 21%). The risk of bleeding did not differ between groups(OR 0.58 [0.15; 2.22] I2 = 86.1%). CONCLUSION: Elevated RPs are significantly associated with increased risk of cardiovascular events and cardiovascular death.

Role of Reticulated Platelets in Cardiovascular Disease.

Human platelets differ considerably with regard to their size, RNA content and thrombogenicity. Reticulated platelets (RPs) are young, hyper-reactive platelets that are newly released from the bone marrow. They are larger and contain more RNA compared to older platelets. In comparison to more mature platelets, they exhibit a significantly higher thrombogenicity and are known to be elevated in patients with an increased platelet turnover such as, diabetics and after acute myocardial infarction. Several studies have shown that RPs correlate with an insufficient antiplatelet response to aspirin and specific P2Y12 receptor inhibitors. In addition, RPs are promising novel biomarkers for the prediction of adverse cardiovascular events in cardiovascular disease. However, the reason for RPs intrinsic hyper-reactivity and their association with ischemic events is not completely understood and the biology of RPs is still under investigation. We here present a structured review of preclinical and clinical findings concerning the role of RPs in cardiovascular disease.

Mass cytometry of platelet-rich plasma: a new approach to analyze platelet surface expression and reactivity.

Mass cytometry (CyTOF) is a new technology that allows the investigation of protein expression at single cell level with high resolution. While several protocols are available to investigate leukocyte expression, platelet staining and analysis with CyTOF have been described only from whole blood. Moreover, available protocols do not allow sample storage but require fresh samples to be obtained, processed, and measured immediately. We provide a structured and reproducible method to stain platelets from platelet-rich plasma to study thrombocyte protein expression and reactivity using mass cytometry. With our method, it is possible to acquire a large number of events allowing deep bioinformatic investigation of platelet expression heterogeneity. Integrated in our protocol is also a previously established freezing protocol that allows the storage of stained samples and to delay their measurement. Finally, we provide a structured workflow using different platelet stimulators and a freely available bioinformatic pipeline to analyze platelet expression. Our protocol unlocks the potential of CyTOF analysis for studying platelet biology in health and disease.

Early detection of elevated cardiac biomarkers to optimise risk stratification in patients with COVID-19.

OBJECTIVE: Risk stratification is crucial to optimise treatment strategies in patients with COVID-19. We aimed to evaluate the impact on mortality of an early assessment of cardiac biomarkers in patients with COVID-19. METHODS: Humanitas Clinical and Research Hospital (Rozzano-Milan, Lombardy, Italy) is a tertiary centre that has been converted to the management of COVID-19. Patients with confirmed COVID-19 were entered in a dedicated database for cohort observational analyses. Outcomes were stratified according to elevated levels (ie, above the upper level of normal) of high-sensitivity cardiac troponin I (hs-TnI), B-type natriuretic peptide (BNP) or both measured within 24 hours after hospital admission. The primary outcome was all-cause mortality. RESULTS: A total of 397 consecutive patients with COVID-19 were included up to 1 April 2020. At the time of hospital admission, 208 patients (52.4%) had normal values for cardiac biomarkers, 90 (22.7%) had elevated both hs-TnI and BNP, 59 (14.9%) had elevated only BNP and 40 (10.1%) had elevated only hs-TnI. The rate of mortality was higher in patients with elevated hs-TnI (22.5%, OR 4.35, 95% CI 1.72 to 11.04), BNP (33.9%, OR 7.37, 95% CI 3.53 to 16.75) or both (55.6%, OR 18.75, 95% CI 9.32 to 37.71) as compared with those without elevated cardiac biomarkers (6.25%). A multivariate analysis identified concomitant elevation of both hs-TnI and BNP as a strong independent predictor of all-cause mortality (OR 3.24, 95% CI 1.06 to 9.93). CONCLUSIONS: An early detection of elevated hs-TnI and BNP predicts mortality in patients with COVID-19. Cardiac biomarkers should be systematically assessed in patients with COVID-19 at the time of hospital admission in order to optimise risk stratification.

MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases.

Reactive oxygen species (ROS) affect many cellular functions and the proper redox balance between ROS and antioxidants contributes substantially to the physiological welfare of the cell. During pathological conditions, an altered redox equilibrium leads to increased production of ROS that in turn may cause oxidative damage. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level contributing to all major cellular processes, including oxidative stress and cell death. Several miRNAs are expressed in response to ROS to mediate oxidative stress. Conversely, oxidative stress may lead to the upregulation of miRNAs that control mechanisms to buffer the damage induced by ROS. This review focuses on the complex crosstalk between miRNAs and ROS in diseases of the cardiac (i.e., cardiac hypertrophy, heart failure, myocardial infarction, ischemia/reperfusion injury, diabetic cardiomyopathy) and pulmonary (i.e., idiopathic pulmonary fibrosis, acute lung injury/acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, lung cancer) compartments. Of note, miR-34a, miR-144, miR-421, miR-129, miR-181c, miR-16, miR-31, miR-155, miR-21, and miR-1/206 were found to play a role during oxidative stress in both heart and lung pathologies. This review comprehensively summarizes current knowledge in the field.

MicroRNA-133 modulates the ß1-adrenergic receptor transduction cascade.

RATIONALE: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate ß-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of ß-adrenergic receptors leads to impaired cardiac function, and ß-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. OBJECTIVE: To determine whether miR-133 affects ß-adrenergic receptor signaling during progression to heart failure. METHODS AND RESULTS: Based on bioinformatic analysis, ß1-adrenergic receptor (ß1AR) and other components of the ß1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective ß1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic ß1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. CONCLUSIONS: miR-133 controls multiple components of the ß1AR transduction cascade and is cardioprotective during heart failure.