Platelets in the NETworks interweaving inflammation and thrombosis

Abstract

Platelets are well characterized for their indispensable role in primary hemostasis to control hemorrhage. Research over the past years has provided a substantial body of evidence demonstrating that platelets also participate in host innate immunity. The surface expression of pattern recognition receptors, such as TLR2 and TLR4, provides platelets with the ability to sense bacterial products in their environment. Platelet α-granules contain microbicidal proteins, chemokines and growth factors, which upon release may directly engage pathogens and/or contribute to inflammatory signaling. Additionally, platelet interactions with neutrophils enhance neutrophil activation and are often crucial to induce a sufficient immune response. In particular, platelets can activate neutrophils to form neutrophil extracellular traps (NETs). This specific neutrophil effector function is characterized by neutrophils expelling chromatin fibres decorated with histones and antimicrobial proteins into the extracellular space where they serve to trap and kill pathogens. Until now, the mechanisms and signaling pathways between platelets and neutrophils inducing NET formation are still not fully characterized. NETs were also detected in thrombotic lesions in several disease backgrounds, pointing towards a role as an interface between neutrophils, platelets and thrombosis, also known as immunothrombosis. The negatively charged DNA within NETs provides a procoagulant surface, and in particular NET-derived proteins may directly activate platelets. In light of the current COVID-19 pandemic, the topic of immunothrombosis has become more relevant than ever, as a majority of COVID-19 patients display thrombi in the lung capillaries and other vascular beds. Furthermore, NETs can be found in the lung and other tissues and are associated with an increased mortality. Here, virus infiltration may lead to a cytokine storm that potently activates neutrophils and leads to massive neutrophil infiltration into the lung and NET formation. The resulting NETs presumably activate platelets and coagulation factors, further contributing to the subsequent emergence of microthrombi in pulmonary capillaries. In this review, we will discuss the interplay between platelets and NETs and the potential of this alliance to influence the course of inflammatory diseases. A better understanding of the underlying molecular mechanisms and the identification of treatment targets is of utmost importance to increase patients' survival and improve the clinical outcome.

Keywords: COVID-19; NETs; immunothrombosis; neutrophil extracellular traps; neutrophils; platelets.

Figure 1

Inflammation-induced platelet-mediated NET formation. The mechanisms leading to platelet-induced NET formation are highly dependent on the surrounding inflammatory conditions. Receptor-ligand binding, as well as soluble mediators, were identified to promote platelet-induced NETosis. Ligation of (soluble) P-sel and neutrophil PSGL-1 was shown to result in NET formation, and also activation of neutrophil Mac-1. SLC44A2 was recently identified as a mechanotransducer of NETosis. CXCR4 and CXCR7, expressed on platelets and neutrophils, may be important to induce NETosis, and functional Adora2b signaling seems indispensable for this. Thrombin-activated platelets not only recruit neutrophils into the inflamed tissue, but also stimulate neutrophils to form NETs. Platelet exosomes, carrying HMGB1 and miRNAs, induce NETosis in a RAGE- and autophagy-dependent manner. TLR9 activation via CpG oligonucleotides lead to the discharge of platelet CXCL4, but CXCL4 also heterodimerizes with CCL5 to induce NETs. Lastly, blocking the neutrophil thromboxane receptor diminished NETosis. CXCR, C-X-C motif chemokine receptor; CXCL4, C-X-C motif ligand 4; CCL5, CC-chemokine ligand 5; GP, glycoprotein; HMGB1, high mobility group box 1; miRNAs, microRNAs; PAR, protease-activated; P-sel, P-selectin; RAGE, receptor for advanced glycation end products; SDF-1, stromal cell-derived factor 1; sP-sel, soluble P-selectin; TLR9, toll-like receptor 9; TXA, thromboxane A; TXA2, thromboxane A₂.

Figure 2

Pathogen-induced platelet-mediated NET formation. Staphylococcus aureus α-toxin induces pore formation in platelets, which then release hBD-1. Gramnegative bacteria and LPS activate platelets in a TLR4-dependent manner, and blocking of LFA-1 in this background abrogated NETosis in neutrophils. Dengue viruses can be sensed by platelets via CLEC2, which leads to shedding of extracellular vesicles promoting NETosis in a TLR2- and CLEC5A-dependent manner. Inlfuenza A virus are recognized via TLR7, and granular C3 then induces NETosis in neutrophils. C3, complement C3; CLEC2, C-type lectin-like receptor 2; CLEC5A, C-type lectin domain containing 5A; hBD-1, human β-defensin 1; ICAM, intercellular adhesion molecule; LFA-1, leukocyte function-associated antigen 1; LPS, lipopolysaccharide; TLR, toll-like receptor.

Figure 3

NETs activate platelets and drive coagulation. Cathepsin G and histones, especially H3 and H4, but also tissue factor directly activate platelets. PtdSer+ve microparticles in NETs provide a negatively charged surface, ideal for accumulation of coagulation factors. NETs with histones activate FXII and FXI, which results in thrombin generation. FXII and FXI also bind PtdSer exposed on platelets. Activated platelets increase P-selectin and GPIIb/IIIa activity, resulting in increased aggregation as well as signaling events leading to release of polyP and dense granule secretion. Ultimately, subsequent thrombin generation leads to clot formation in the vasculature. PAD4 was shown to inhibit ADAMTS13 activity, which hampers cleavage and removal of VWF strings. NET cathepsin G triggers tissue factor production in endothelial cells, further driving coagulation. ADAMTS13, a disintegrin and metalloproteinase with thrombospondin type-1 motif-1, member 13; FXII, factor XII; FXI, factor XI; GP, glycoprotein; MPs, microparticles; PAD4, peptidylarginine deiminase 4; PtdSer, phosphatidylserine; P-sel, P-selectin; polyP, inorganic polyphosphate; VWF, von Willebrand factor.

Figure 4

The vicious cycle of platelet-neutrophil interplay contributing to immunothrombosis in COVID-19. SARS-CoV-2 infection results in a drastic increase in pro-inflammatory cytokines and complement C5a, the occurrence of LDGs and a reduction in endogenous DNases. These effects, as well as the virus itself (via currently unknown mechanisms) may trigger and/ or enhance NET formation. Autoantibodies may further stabilize the formed NETs and protect them against removal. NETs in COVID-19 patients were found to have incorporated procoagulant tissue factor. Platelets are activated by the NETs, but also by an increase in eATP and eADP. Platelets might then react with the production of PtdSer+ve microparticles, which in turn may trigger NETosis and drive coagulation. Endothelial cells also release microparticles, stimulating neutrophils for NET formation. The resulting immunothrombosis very likely contributes to significant end-organ damage. C5a, complement C5a; COVID-19, corona virus disease 2019; DNase, deoxyribonuclease; eADP, extracellular adenosine diphosphate; eATP, extracellular adenosine triphosphate; G-CSF, granulocyte-colony stimulating factor; IL, interleukin; LDG, low density granulocyte; NET, neutrophil extracellular trap; PtdSer+ve, phosphatidylserine positive; TF, tissue factor; TNFα, tumor necrosis factor α; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2.