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.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing...

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Coagulation factor (F) Xa induces proinflammatory responses through activation of protease-activated receptors (PARs). However, the effect of FXa on cardiac fibroblasts (CFs) and the contribution of PARs in FXa-induced cellular signalling in CF has not been fully characterised. To answer these questions, human and rat CFs were incubated with FXa (or TRAP-14, PAR-1 agonist). Gene expression of pro-fibrotic and proinflammatory markers was determined by qRT-PCR after 4 and 24 h. Gene silencing of F2R (PAR-1) and F2RL1 (PAR-2) was achieved using siRNA. MCP-1 protein levels were measured by ELISA of FXa-conditioned media at 24 h. Cell proliferation was assessed after 24 h of incubation with FXa ± SCH79797 (PAR-1 antagonist). In rat CFs, FXa induced upregulation of Ccl2 (MCP-1; >30-fold at 4 h in atrial and ventricular CF) and Il6 (IL-6; ±7-fold at 4 h in ventricular CF). Increased MCP-1 protein levels were detected in FXa-conditioned media at 24 h. In human CF, FXa upregulated the gene expression of CCL2 (>3-fold) and IL6 (>4-fold) at 4 h. Silencing of F2R (PAR-1 gene), but not F2RL1 (PAR-2 gene), downregulated this effect. Selective activation of PAR-1 by TRAP-14 increased CCL2 and IL6 gene expression; this was prevented by F2R (PAR-1 gene) knockdown. Moreover, SCH79797 decreased FXa-induced proliferation after 24 h. In conclusion, our study shows that FXa induces overexpression of proinflammatory genes in human CFs via PAR-1, which was found to be the most abundant PARs isoform in this cell type.