Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism.

No abstract

Summary Antiplatelet drugs targeting G-protein-coupled receptors (GPCRs), used for the secondary prevention of arterial thrombosis, coincide with an increased bleeding risk. Targeting ITAM-linked receptors, such as the collagen receptor glycoprotein VI (GPVI), is expected to provide a better antithrombotic-hemostatic profile. Here, we developed and characterized an ultra-high-throughput (UHT) method based on intracellular [Ca 2+ ] i increases to differentiate GPVI and GPCR effects on platelets. In 96-, 384-, or 1,536-well formats, Calcium-6-loaded human platelets displayed a slow-prolonged or fast-transient [Ca 2+ ] i increase when stimulated with the GPVI agonist collagen-related peptide or with thrombin and other GPCR agonists, respectively. Semi-automated curve fitting revealed five parameters describing the Ca 2+ responses. Verification of the UHT assay was done with a robustness compound library and clinically relevant platelet inhibitors. Taken together, these results present proof of principle of distinct receptor-type-dependent Ca 2+ signaling curves in platelets, which allow identification of new inhibitors in a UHT way.

No abstract

Platelets within one individual display heterogeneity in reactivity, size, age, and expression of surface receptors. To investigate the combined intra-individual contribution of platelet size, platelet age, and receptor expression levels on the reactivity of platelets, we studied fractions of large and small platelets from healthy donors separated by differential centrifugation. Size-separated platelet fractions were perfused over a collagen-coated surface to assess thrombus formation. Multicolour flow cytometry was used to characterise resting and stimulated platelet subpopulations. Platelet age was determined based on RNA and HLA-I labelling. Signal transduction was analysed by measuring consecutive phosphorylation of serine/threonine-protein kinase Akt. Large platelets adhered faster to collagen under flow and formed larger thrombi, compared to small platelets. Among the large platelets a highly reactive juvenile platelet subpopulation was identified with high GPVI expression. Elevated GPVI expression correlated with high HLA-I expression, RNA content and increased platelet reactivity. Akt phosphorylation and activation upon collagen stimulation differed stronger between juvenile and older platelets than between large and small platelets. GPVI expression and platelet reactivity decreased throughout platelet storage at 22°C and was better maintained throughout cold storage at 4°C. We further detected a higher GPVI expression in platelets of patients with immune thrombocytopenia. Our findings show that high GPVI expression is a feature of highly reactive juvenile platelets, which are predominantly found among the large platelet population explaining the better performance of large platelets during thrombus formation. These data are important for studies of thrombus formation, platelet storage and ITP.