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.

JCAD promotes arterial thrombosis through PI3K/Akt modulation: a translational study.

AIMS: Variants of the junctional cadherin 5 associated (JCAD) locus associate with acute coronary syndromes. JCAD promotes experimental atherosclerosis through the large tumor suppressor kinase 2 (LATS2)/Hippo pathway. This study investigates the role of JCAD in arterial thrombosis. METHODS AND RESULTS: JCAD knockout (Jcad-/-) mice underwent photochemically induced endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with JCAD small interfering RNA (siJCAD), LATS2 small interfering RNA (siLATS2) or control siRNA (siSCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome or ST-elevation myocardial infarction (STEMI). Jcad-/- mice displayed reduced thrombogenicity as reflected by delayed time to carotid occlusion. Mechanisms include reduced activation of the coagulation cascade [reduced tissue factor (TF) expression and activity] and increased fibrinolysis [higher thrombus embolization episodes and D-dimer levels, reduced vascular plasminogen activator inhibitor (PAI)-1 expression]. In vitro, JCAD silencing inhibited TF and PAI-1 expression in HAECs. JCAD-silenced HAECs (siJCAD) displayed increased levels of LATS2 kinase. Yet, double JCAD and LATS2 silencing did not restore the control phenotype. si-JCAD HAECs showed increased levels of phosphoinositide 3-kinases (PI3K)/ proteinkinase B (Akt) activation, known to downregulate procoagulant expression. The PI3K/Akt pathway inhibitor-wortmannin-prevented the effect of JCAD silencing on TF and PAI-1, indicating a causative role. Also, co-immunoprecipitation unveiled a direct interaction between JCAD and Akt. Confirming in vitro findings, PI3K/Akt and P-yes-associated protein levels were higher in Jcad-/- animals. Lastly, as compared with chronic coronary syndrome, STEMI patients showed higher plasma JCAD, which notably correlated positively with both TF and PAI-1 levels. CONCLUSIONS: JCAD promotes arterial thrombosis by modulating coagulation and fibrinolysis. Herein, reported translational data suggest JCAD as a potential therapeutic target for atherothrombosis.

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.

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.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.