Platelet-leukocyte crosstalk in COVID-19: How might the reciprocal links between thrombotic events and inflammatory state affect treatment strategies and disease prognosis?

Abstract

Platelet-leukocyte crosstalk is commonly manifested by reciprocal links between thrombosis and inflammation. Platelet thrombus acts as a reactive matrix that recruits leukocytes to the injury site where their massive accumulation, activation and migration promote thrombotic events while triggering inflammatory responses. As a life-threatening condition with the associations between inflammation and thrombosis, COVID-19 presents diffuse alveolar damage due to exaggerated macrophage activity and cytokine storms. These events, together with direct intracellular virus invasion lead to pulmonary vascular endothelialitis, cell membranes disruption, severe endothelial injury, and thrombosis. The developing pre-alveolar thrombus provides a hyper-reactive milieu that recruits circulating leukocytes to the injury site where their activation contributes to thrombus stabilization and thrombosis propagation, primarily through the formation of Neutrophil extracellular trap (NET). NET fragments can also circulate and deposit in further distance where they may disseminate intravascular thrombosis in severe cases of disease. Thrombi may also facilitate leukocytes migration into alveoli where their accumulation and activation exacerbate cytokine storms and tissue damage, further complicating the disease. Based on these mechanisms, whether an effective anti-inflammatory protocol can prevent thrombotic events, or on the other hand; efficient antiplatelet or anticoagulant regimens may be associated with reduced cytokine storms and tissue damage, is now of interests for several ongoing researches. Thus shedding more light on platelet-leukocyte crosstalk, the review presented here discusses the detailed mechanisms by which platelets may contribute to the pathogenesis of COVID-19, especially in severe cases where their interaction with leukocytes can intensify both inflammatory state and thrombosis in a reciprocal manner.

Keywords: Anti-inflammatory agents; Antiplatelet drugs; COVID-19; Cytokine storms; Damage-associated molecular pattern molecules; Leukocyte Migration; NETs; Platelets; Thrombosis.

Fig. 1

Platelet leukocyte crosstalk in COVID-19 a mutual link between thrombosis and inflammation. A) Alveolar phase: SARS-CoV-2 invasion to type II peneumocyte induces cytokine release (1). These cytokines and the direct interaction of the virus with tissue-resident monocyte/macrophage turn them into a hyper-inflammatory phenotype with exaggerated activity that contributes to cytokine storms and tissue damage (2). The crosstalk between activated macrophage and other immune cells supports a robust “cytokine storm syndrome” in severe case of disease (3). On the one hand, under the influence of such an inflammatory milieu, exhausted NK cells (4) fail to eradicate infected macrophage, while on the other hand, the infected cells that express lower MCH class I, also evade from recognition by cytotoxic T cells. Under such a condition, hyper-activated macrophages freely contribute to tissue damage and vascular injury either by releasing ROS (5) and other toxic agents or by inducing NETs formation (6). These are events that link COVID-19-induced inflammatory responses to vascular thrombotic events. B) Intravascular phase: Endothelitis due to direct invasion of SARS-CoV-2 or indirect damage by macrophages and hyperinflammatory condition may cause injury in adjacent alveolar vessels where the platelets recruitment creates a reactive thrombus attracting leukocytes (1). The recruited leukocytes may migrate to the alveolar tissue through the thrombus (2), or release the extracellular traps in a low shear pocket that propagate thrombosis (3). Cytokine milieu orchestrated by hyper-inflamed or damaged alveolar tissue, may also urge adjacent endothelium to express inflammatory molecules and receptors, which recruit circulating leukocytes (4). Produced NETs on endothelium or thrombi may be released from their original site and disseminated while their deposition at greater distances can create secondary foci of thrombosis (5). In severe condition of disease, blood-borne viruses may also directly affect circular platelets and leucocytes while causing their activation (6). Abbreviations: Th1: T-helper cell type 1; NK: natural killer cell; TC: cytotoxic T cell; MQ: macrophage; NET: neutrophil extracellular trap; ROS: reactive oxygen species.

Fig. 2

Different Pathways of platelet activation in COVID-19. A) Direct interaction of virus with platelets: The SARS-CoV-2 - may engage with platelet receptors such as ACE2 and CD147 and activate various intracellular signaling pathways. Platelets also endocytose SARS-CoV-2 or its RNA, creating an inflammasome in which the AKT and P38-MAPK pathways are activated when SARS-CoV-2 RNA interacts with TLR7. Followed by TLR7 engagement with virus RNA, an adaptor protein, MYD88, activates other adaptor proteins such as IRAK1, IRAK4, TRAF6 (that activate AKT and P38-MAPK pathways), and TAK1, IKK, SNAP-23 (that lead to C3 release and C3-induced NETosis). Generally, these activating pathways lead to enhanced expression of P-selectin and CD40L, increased platelet-leukocyte interactions and PLAs formation. B). The effect of cytokine storms on platelets: Overexpression of inflammatory cytokines such as IL-6, IL-1, and TNF-α may activate jak2/STAT3 and its cross-talk with GPVI/collagen signaling, P38/MAPK, and NF-κB, respectively, leading to platelet hyperactivation. C) SARS-CoV-2-induced tissue/endothelial damage: DAMPs and mt-DNA released by damaged tissues and endothelial cells are sensed with TLR4 and TLR9 and activate NF-κB/PKC-ERK and IRAK1/PI3K-AKT respectively, leading to platelet activation D). Leukocyte-induced platelet activation: Leukocytes may activate platelets through their released mediators. Here, in a conserved milieu, leucocyte derived NET components and cathelicidin (interacting with TLR2, 4, and GPVI, respectively), chemokines/chemoattractants (particularly PAF interacting with GPCRs) as well as ROS and proteolytic enzymes (that induce the generation of fibrin interacting with GPVI), promote platelet activation and pro-coagulant function. Abbreviations: ACE2: Angiotensin-converting enzyme-2; AKT: protein kinase B; GPCRs: G protein–coupled receptors; IKK: IκB kinase; IRAK: Interleukin-1 receptor-associated kinase; JAK: Janus kinase; JNK: c-Jun N-terminal kinase (a major signaling cassettes of MAPK signaling pathway); MAPK: mitogen-activated protein kinase (MAPK) MYD88: Myeloid differentiation primary response 88; NET: neutrophil extracellular trap; NF-κB: nuclear factor-kB; PAF: platelet activating factor; PLA: platelet-leukocyte aggregations; PLC: phospholipase C; PKC: Protein kinase C; ROS: reactive oxygen species; SNAP-23: Synaptosomal-associated protein 23; Src: sarcoma virus tyrosine kinase; SYK: spleen tyrosine kinase; TAK: transforming growth factor-β-activated kinase; TLR: toll like receptor; TNF: tumor necrosis factor; TRAF: Tumor necrosis factor receptor–associated factor.

Fig. 3

Thrombus effectively gates inflammatory leukocytes into the alveolar space. A) Evidence for efficient leukocyte migration through thrombi. ai) Representative images depicting polarized and spread leukocytes (Gr-1 Ab, green) on C57Bl/6 mouse mesenteric veins after IR injury in mesenteric vasculature. As depicted, area with aggregated platelets were at least 20-fold more effective in inducing leukocyte shape change and migration compared to where leukocytes directly interacting with the endothelium free of platelets (Ghasemzadeh et al. Blood 2013). aii) Representative image at 10 min post injury (needle injury on mesenteric veins) depicting migrating leukocytes (Gr-1 Ab, green) at different positions between the margin and the center of a thrombus (platelet stained with DiOC6 in red). aiii) GFP-NOD mice were injected systemically with an anti Gr-1 Ab (for leukocyte staining in red), prior to induction of vascular injury via needle puncture. Thrombus formation (in yellow greenish) was monitored by confocal microscopy where the confocal sections through the “Top”, “Middle” and “Base” confirmed directed intravascular leukocyte migration into thrombi (Ghasemzadeh et al. Thrombosis & Hemostasis 2015). B) Thrombi facilitate directed migration of circulating leucocytes from blood to infected alveolar tissue dictated by the cytokine gradient. (a) Alveolar vessel injured by direct invasion of SARS-CoV-2 or fully agitated macrophage under hyper-inflammatory state, recruits platelets that forms a developing thrombus at site of injury. (b) P-selectin expressing platelets on thrombus recruit leukocytes while their activation in pro-inflammatory milieu leads to cytokine-induced arrest and their subsequent adhesion and activation. Fully activated neutrophils may release NET that propagates thrombosis (c) or migrate to the source of inflammation through the thrombi (d), in a direction that is dictated by continuous release of cytokines/chemo-attractants (showing by blue dots) from the site of infection and alveolar damage. Abbreviations: Ab: antibody; IR: ischemia-reperfusion; NET: neutrophil extracellular traps. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)