To Gain Insights into the Pathophysiological Mechanisms of the Thrombo-Inflammatory Process in the Atherosclerotic Plaque

Abstract

Thromboinflammation, the interplay between thrombosis and inflammation, is a significant pathway that drives cardiovascular and autoimmune diseases, as well as COVID-19. SARS-CoV-2 causes inflammation and blood clotting issues. Innate immune cells have emerged as key modulators of this process. Neutrophils, the most predominant white blood cells in humans, are strategically positioned to promote thromboinflammation. By releasing decondensed chromatin structures called neutrophil extracellular traps (NETs), neutrophils can initiate an organised cell death pathway. These structures are adorned with histones, cytoplasmic and granular proteins, and have cytotoxic, immunogenic, and prothrombotic effects that can hasten disease progression. Protein arginine deiminase 4 (PAD4) catalyses the citrullination of histones and is involved in the release of extracellular DNA (NETosis). The neutrophil inflammasome is also required for this process. Understanding the link between the immunological function of neutrophils and the procoagulant and proinflammatory activities of monocytes and platelets is important in understanding thromboinflammation. This text discusses how vascular blockages occur in thromboinflammation due to the interaction between neutrophil extracellular traps and ultra-large VWF (von Willebrand Factor). The activity of PAD4 is important for understanding the processes that drive thromboinflammation by linking the immunological function of neutrophils with the procoagulant and proinflammatory activities of monocytes and platelets. This article reviews how vaso-occlusive events in thrombo-inflammation occur through the interaction of neutrophil extracellular traps with von Willebrand factor. It highlights the relevance of PAD4 in neutrophil inflammasome assembly and neutrophil extracellular traps in thrombo-inflammatory diseases such as atherosclerosis and cardiovascular disease. Interaction between platelets, VWF, NETs and inflammasomes is critical for the progression of thromboinflammation in several diseases and was recently shown to be active in COVID-19.

Keywords: COVID-19; NETosis; neutrophil extracellular traps; neutrophil inflammasome; thromboinflammation; ultra-large VWF.

Figure 1

Platelet-endothelium adhesion occurs when activated endothelial surfaces express P-selectin, which interacts with platelet surface receptors GPIbα and PSGL-1 to mediate platelet rolling. Beta-3 integrins subsequently mediate firm adhesion. From Gawaz et al. Ref. [8].

Figure 2

Neutrophil activation is mediated by several factors including IL-8, G-CSF, resistin, lipocalin-2, hepatocyte growth factor and NET re-release. The immune responses of both NK and T lymphocytes contribute to the formation of NETs, which in turn activate the complete system (C5 and C3). This leads to microvascular thrombosis and subsequent organ damage. Abbreviations: C, complement; GF, growth factor; IL, interleukin; NK, natural killer. Other abbreviations are listed in the previous figure. From Ref. [26].

Figure 3

The formation of Neutrophil Extracellular Traps (NETs) in severe COVID-19 patients leads to cardiac injury caused by vascular inflammation, thrombogenesis, and NETOSIS, which arise from the unstable atherosclerotic plaque. Abbreviations used: HMGB1, High Mobility Group Box 1; ISG-15, Interferon-Stimulated Gene 15; LDG, Low-Density Granulocytes; NDG, Normal Density Granulocytes; NAD, Nicotinamide Adenine Dinucleotide; ROS, Reactive Oxygen Species; SIRT3, Sirtuin 3. The same abbreviations are used as in the previous figure. The symbol ↑ represents an increase, while ↓ represents a decrease. From Ref. [57].

Figure 4

Flow models were used to investigate interactions between von Willebrand factor (VWF) and neutrophils, providing insights into the relationships between the A1 domain of VWF multimers, platelets, neutrophils, and NETs under conditions of high shear flow (indicated by red arrows) and low shear flow (indicated by blue arrows). Abbreviations; ds, double strand; GP, glycoprotein.

Figure 5

Typical signalling pathways for NLRP3 inflammasome activation. Upon stimulation of TLR4, IL-1R, or TNFR, TNF receptor-associated factor 2 (TRAF2) and TNF receptor-associated factor 6 (TRAF6) recruit the inhibitor of nuclear factor-κB kinase α/β (IKKα/β), resulting in the translocation of NF-κB subunits to the nucleus. This enhances the transcription of NLRP3 and pro-IL-1β, facilitating the activation of components in the local systems. Upon NLRP3 inflammasome initiation through PAMPs and DAMPs, the subunits of NLRP3 and pro-IL-1β are enabled. As a result, the inactive procaspase-1 is cleaved into active caspase-1, which triggers the following assembly of NLRP3 inflammasome. Once activated, the NLRP3 inflammasome initiates the processing of signals. Gasdermin D, pro-IL-1β, and pro-IL-18 are transformed into their biologically active forms through the cleavage of dormant procaspase-1 into active caspase-1, which then triggers the processing of gasdermin D, pro-IL-1β, and pro-IL-18 into their biologically active forms. From Wu et al. Ref. [123].

Figure 6

Inflammatory pathways implicated in atherosclerosis are illustrated. Preclinical and clinical trials have revealed a complex equilibrium between pro-inflammatory and anti-inflammatory pathways. The balance is regulated by inflammatory cells (macrophages and T-cells) and the liver (CRP), promoting endothelial dysfunction and the progression of atherosclerotic plaque via the production of molecules with either a pro-inflammatory (red box) or anti-inflammatory (blue box) effect. Plaque rupture is a potential result of an intensified inflammatory process. Abbreviations used: CRP (C-reactive protein); MMPs (matrix metalloproteinases); IFN-γ (interferon-gamma); IL-1α (interleukin-1-alpha); IL-1β (interleukin-1-beta); IL-2 (interleukin-2); IL-6 (interleukin-6); IL-10 (interleukin-10). IL-18, interleukin-18; Lp-PLA2, lipoprotein-associated phospholipase A2; TGF-β, transforming growth factor beta; Th-1, T-helper-1 lymphocyte; TNF-α, tumor necrosis factor alpha; and T-reg, regulatory T lymphocyte. From Ref. [146].

Figure 7

NETs cause arterial blockage. During atherothrombosis, NETs produced by neutrophils can contribute to a range of biochemical events that can activate the coagulation process. By destabilising the atheromatous plaque and inducing its rupture, NETs enhance the stability of the blood clot. From Ref. [2].