Platelets and viruses

Abstract

Platelets play an essential role in maintaining vascular integrity after injury. In addition, platelets contribute to the immune response to pathogens. For instance, they express receptors that mediate binding of viruses, and toll-like receptors that activate the cell in response to pathogen-associated molecular patterns. Platelets can be beneficial and/or detrimental during viral infections. They reduce blood-borne viruses by engulfing the free virus and presenting the virus to neutrophils. However, platelets can also enhance inflammation and tissue injury during viral infections. Here, we will discuss the roles of platelets in viral infection.

1. Introduction

Platelets are released by megakaryocytes in large numbers into the blood.¹ The main function of these small anucleate cells is to maintain vascular integrity after injury of the vasculature. Uncontrolled platelet activation can also lead to intravascular thrombosis. Platelets contain receptors that mediate adhesion to the damaged vessel wall, such as glycoprotein (GP) Ib-V-IX, GPVI and α2β1, and receptors that respond to soluble agonists, such as P2Y12, thromboxane receptor and protease-activated receptor (PAR) 1 and 4.² Platelets contain three different types of granule. Dense (δ) granules contain mediators that regulate vascular tone, such as nucleotides (e.g. ADP and GTP), bioactive amides (e.g. histamine and serotonin) and bioactive ions (e.g. Ca²⁺ and PO₃⁻). Alpha (α) granules contain proteins that can be classified into five groups: adhesion molecules, platelet microbicidal proteins and kinocidins, mitogenic factors, coagulation factors and protease inhibitors. Finally, lysosomal (λ) granules contain enzymes, such as proteases and glycosidases, that can modulate platelet-fibrin retraction needed for wound healing.³ A variety of platelet inhibitors, including acetylsalicylic acid (ASA) and P2Y12 receptor antagonists, are used to prevent arterial thrombosis.²

1.1. Immune functions of platelets

Platelets play a role in the immune response to pathogens.⁴ They express a variety of receptors, including lectins, integrins, and toll-like receptors (TLRs), that mediate pathogen binding and cellular activation (Figure).^5–9 In addition, human platelets express Fc receptor FcγRIIA, which recognize immune complexes.¹⁰ Infections are often associated with thrombocytopenia due to increased platelet activation.^{8, 9} Activated platelets release extracellular vesicles (EVs) that express CD62P (P-selectin) and contain an array of chemokines, such as CCL5 (RANTES), and cytokines, such as IL-1β.¹¹ Activated platelets also bind to leukocytes, including monocytes, neutrophils, eosinophils and lymphocytes. This binding is mediated by various receptor-receptor pairs, such as platelet CD62P and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1) and platelet CD40 and leukocyte CD154 (CD40 ligand, CD40L) and platelet GPIbα and leukocyte MAC-1 (CD11b/CD18).¹¹ Platelet binding induces gene expression and cellular response in the leukocytes.^{11, 12} Platelet-leukocyte aggregates are used as a marker of platelet activation in infectious diseases. Moreover, platelet binding to neutrophils and eosinophils can induce the formation of extracellular traps, such as neutrophil extracellular traps (NETs).^11–16 This extracellular DNA immobilizes pathogens but can also contribute to thrombosis and tissue damage.^{17, 18} Platelets modulate natural killer (NK) cell activity by either direct interaction or indirectly by releasing mediators, including TGFβ and EVs.^{19, 20} Platelets also facilitate the recruitment of a variety of inflammatory cells, including neutrophils, cytotoxic T cells and dendritic cells, to sites of inflammation.^{21, 22} Platelet-derived CXCL4 (platelet factor 4, PF4), CCL5 and serotonin activate T cells.²³ Platelets bind to and activate dendritic cells through JAM C-Mac1, CD62P-PSGL-1 and CD40-CD154 interactions.^{11, 24} Finally, platelets contribute to adaptive immunity by increasing IgG1, IgG2 and IgG3 production by B cells.^{6, 11}

Figure. Platelet receptors which mediate virus-platelet interaction and initiate intracellular signaling events.

HIV and DENV bind to CLEC-2 and DC-SIGN. DENV binds to GPIb and human CMV (hCMV) interacts with TLR2. Depending on the virus, HIV, HCV, DENV, IAV, EMCV and CVB3 can be detected depending on their genomic state as ssRNA, dsDNA or as dsRNA directly or as genomic intermediate during replication of ssRNA viruses by endosomal expressed TLRs. TLR7/8 detect ssRNA, TLR3 dsRNA and TLR9 dsDNA. Figure created in BioRender.com.

1.2. Platelets and viruses

A variety of viruses, including enteroviruses [Coxsackievirus, CVB; encephalomyocarditis virus, EMCV], influenza A viruses (IAV), coronaviruses (CoV), dengue virus (DENV), human immunodeficiency virus type-1 (HIV-1), hepatitis C virus (HCV), herpes [herpes simplex virus type 1, HSV-1; cytomegalovirus, CMV; vaccinia virus], and adenoviruses, have been shown to bind to and activate platelets (Figure).^{9, 11, 25} Larger DNA viruses from the herpes viral family, such as HSV-1, bind to platelets but are not internalized.^26–28 Binding of human CMV to TLR2 on platelets triggers platelet binding to neutrophils as well as to T cells, B cells and dendritic cells.²⁸ In contrast, smaller RNA viruses, such as CVB, EMCV, DENV, IAV, HIV-1, and HCV, bind to platelets and are internalized.^{6, 29–33} HIV-1 and DENV bind to two C-type lectins called dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and C-type lectin-like receptor 2 (CLEC-2) (Figure).^{34, 35} In addition, DENV binds to GPIb.³⁶ Interestingly, DENV replicates in platelets.³⁴ A study found that DENV and H5N1 IAV activation of CLEC-2 increases release of EVs.¹³ Immune complexes formed during infection, such as IAV, can activate human platelets via platelet FcγRIIA signaling.¹⁰

Platelets express all 10 TLRs.^{37, 38} They recognize viral single-stranded (ss) RNA or double-stranded (ds) RNA via the intracellular receptors TLR7/8 or TLR3, respectively (Figure). DsRNA is a by-product of ssRNA virus replication. Stimulation of platelet TLR7 leads to cell surface expression of CD62P and formation of platelet-neutrophil aggregates whereas TLR3 stimulation does not induce CD62P surface expression but increases platelet responsiveness to thromboxane, ADP or collagen stimulation.^{11, 30, 39} In addition, platelets express TLR9 which detects dsDNA from DNA viruses.⁴⁰

Infection with RNA viruses often induces thrombocytopenia. For instance, DENV infection in humans lead to thrombocytopenia and hemorrhagic fever. DENV infection of rhesus macaques led to the formation of platelet-monocyte aggregates at 24 hours and platelet-neutrophils aggregates 3 days post infection.⁴¹ Platelet-monocyte aggregates are also increased during HIV and IAV infection.^{42, 43} H1N1 IAV binding to platelets also induces release of EVs.¹⁰ Parvovirus B19 infection in humans is associated with increased levels of circulating platelet-derived EVs.⁴⁴ However, parvovirus B19 infection in mice did not increase platelet EV levels in vivo.⁴⁴ EMCV infection of mice leads to the formation of platelet-neutrophil aggregates 1 hour post infection and thrombocytopenia 24 hours post infection.³⁰ In a mouse model of CVB3 infection, platelets internalize CVB3 and express phosphatidylserine and CD62P on their surface and bind to neutrophils.⁶ However, CVB3 does not replicate in platelets.⁶

Platelets can be beneficial or detrimental to the host during virus infection.^{6, 45, 46} In this review we will focus on the role of platelets in virus infection of the heart and lung.

2. Platelets and virus infection of the heart

Viral infections can result in myocarditis, which is a leading cause of sudden cardiac death in children and young adults.⁴⁷ Indeed, several different viruses, including enteroviruses, adenoviruses, herpesviruses, HIV, parvovirus B19, and IAV, have been detected in endomyocardial biopsies. CVB3 and EMCV are known to induce viral myocarditis in humans and animals (Table 1). Clinical observations and animal studies showed that enteroviral virus infection causes thrombocytopenia.^{6, 30, 48, 49} One study showed that either injection of a TLR7 agonist into mice or EMCV infection of mice led to thrombocytopenia that was abolished in TLR7 knockout mice.³⁰ However, TLR7 stimulation did not initiate a prothrombotic phenotype in platelets.³⁰ In a mouse model of CVB3 infection, depletion of platelets led to increased levels of CVB3 and a reduced adaptive immune response.⁶ In a mouse model of EMCV infection, depletion of platelets increased mortality.³⁰ These studies indicate that platelets are protective by limiting viremia and contributing to long lasting immunity. Interestingly, we found that CD62P deficiency resulted in increased CVB3 myocarditis in mice, which may be due to decreased platelet-leukocyte binding and removal of virus infected platelets.⁵⁰

Table 1:

Role of platelets in viral infection of the heart

Pathogen	Observation	References
CVB3	Platelets engulf CVB3 and limit viremia	6
CVB3	Infection leads to platelet activation	6
EMCV	Platelet TLR7 recognizes EMCV which leads to platelet activation	30
CVB3, EMCV	Infection associated with thrombocytopenia	6, 30, 48, 49
CVB3, EMCV	Platelets limit overall virus infection	6, 30
CVB3, EMCV	Platelet-neutrophil aggregates enhance viral clearance	6, 30
CVB3	Platelet depletion affects adaptive immune responses	6
CVB3	Platelet depletion results in more severe myocarditis and increased mortality	6

Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) infection and Coronavirus disease 2019 (COVID-19) is associated with cardiac injury suggesting that the virus directly affects the heart.⁵¹ Indeed, viral genomes have been detected in the hearts of COVID-19 patients with myocarditis and heart failure.^52–54 At present, the role of platelets in the cardiac pathology associated with COVID-19 is not known.

3. Platelets and respiratory virus infection

There are a variety of viruses that infect the lung (Table 2). For instance, seasonal flu is a recurring public health threat causing serious morbidity and mortality in the young and elderly populations.⁵⁵ Infection with respiratory viruses, such as IAV or respiratory syncytial virus (RSV) infections, is associated with an increased incidence of myocardial infarction.⁵⁶ IAV infection is also associated with venous thromboembolism.⁵⁷ Thrombocytopenia was shown to be a risk factor for mortality in acute IAV infection.⁵⁸ Platelets are activated during IAV infection directly by locally generated agonists or damage signals.^{10, 59} In addition, H1N1 IAV immune complexes directly activates platelets through FcγRIIA.¹⁰ Furthermore, H1N1 IAV-platelet interaction increases GPIIb/IIIa activation, platelet thromboxane signaling and EV release.¹⁰

Table 2:

Role of platelets in respiratory virus infection

Pathogen	Observation	References
IAV, RSV, SARS-CoV-2	Increased risk of cardiovascular events including myocardial infarction or venous thrombosis	56, 57, 63–65
IAV, SARS-CoV-2	Platelets can engulf viruses and limit viremia	15, 68, 69
IAV, SARS-CoV-2	Formation of platelet-monocyte, platelet-neutrophil and platelet-T cell aggregates	16, 42, 67, 68
IAV	Platelets bind to endothelial cells	60
IAV, SARS-CoV-2	Platelets enhance NET formation	13, 15, 17, 18, 66
H1N1	Directly activates platelets via FcγRIIA	10
H1N1, SARS-CoV-2	Virus-platelet interaction increases platelet activation and EV release	10, 59, 67, 68
H5N1	Binding to CLEC-2 enhances platelet EV release	13
H1N1	Accumulation of platelets in lung correlates with CD62P expression and disease severity	16, 45, 61
IAV	Thrombocytopenia during IAV infection predicts increased mortality	58
H1N1	Anti-platelet drugs (e.g. ASA or clopidogrel) improve outcome in infected mice	16, 45, 46, 60
SARS-CoV-2	Megakaryocytes in capillaries of lungs and hearts	70, 71

Human platelets can engulf IAV in vitro and patients infected with IAV have viral particles within platelets.¹⁵ In mouse models, accumulation of platelets within the lung correlated with IAV disease progression.^{16, 45} Early after IAV infection platelets form heterotypic aggregates with monocytes.⁴² Platelet-neutrophil aggregates were shown to transmigrate from the circulation into the airspace of IAV infected mouse lungs.¹⁶ In vitro studies showed that platelets attach to IAV infected endothelial cells via integrin mediated mechanisms which can be reduced by anti-platelet drugs.⁶⁰ Importantly, administration of the anti-platelet drugs ASA and clopidogrel reduced IAV-induced lung injury in mice.^{16, 45, 60} We confirmed that ASA improved the survival of mice infected with H1N1 IAV.⁴⁶ Levels of CD62P increased in the lungs of IAV infected mice, which correlated with increased platelet numbers in the lung.^{45, 61} In addition, clopidogrel was shown to reduce levels of CD62P but did not IL-6 or CXCL1 in the lungs of IAV infected mice.¹⁶ We observed that CD62P deficiency in mice was associated with increased survival after IAV infection compared to wild-type controls.⁵⁰ These studies indicate that platelets contribute to lung injury in a mouse model of IAV infection possible by increasing inflammation, neutrophil recruitment and NET formation.^{17, 18} One study showed that IAV infection of human platelets led to engulfment of the virus, activation of TLR7 and expression of C3, which trigger neutrophils to release NETs.¹⁵

CoVs, including HCoVs 229E, NL63, OC43 and HKU1, are among the causative agents of the common cold that usually infect the nose and upper respiratory tract.⁶² However, more severe disease is linked to emerging CoV strains: SARS-CoV, Middle East respiratory syndrome (MERS) CoV and SARS-CoV-2. SARS, MERS and COVID-19 are associated with thrombocytopenia in humans and mice.⁶³ Reduced platelet count is associated with increased morbidity and mortality in pandemic CoV infections.⁶³ COVID-19 patients also have an increased risk of thrombosis.^{64, 65}

The potential role of platelets in COVID-19 was recently discussed.⁶⁶ As expected, two studies recently reported platelet activation in COVID-19 patients.^{67, 68} Platelets from COVID-19 patients had increased levels of CD62P and platelet-neutrophil, platelet-leukocyte and platelet-T cell aggregates numbers were increased.⁶⁸ Similarly, a second study found increased levels of platelet-monocyte aggregates in severe COVID-19 patients.⁶⁷ Two studies have also shown platelets from COVID-19 patients contain SARS-CoV-2 mRNA, which indicates that platelets can take up viral mRNA.^{68, 69} Interestingly, postmortem studies observed megakaryocytes in capillaries of lungs and hearts of COVID-19 patients.^{70, 71} Importantly, megakaryocytes are present in the lung and have immunomodulatory activity.^{1, 72, 73} In addition, neutrophil-mediated thrombo-inflammation is a feature of COVID-19 and associated with disease severity with increased levels of NETs in pulmonary vessels and in the circulation.^{4, 74–82}

One study showed that SARS-CoV-2 infection leads to changes in platelet gene expression.⁶⁸ These changes were similar to the pattern observed in platelets from pandemic H1N1 IAV infected patients, suggesting a common anti-viral response in platelets after viral infection.⁶⁸ Platelets from COVID-19 patients expressed increased levels of the antiviral protein interferon induced transmembrane protein 3 (IFITM3).⁶⁸ Expression of a nonfunctional IFITM3 variant correlates with COVID-19 severity.⁸³ In mouse studies, IFITM3 was shown to protect against IAV infection.^{84, 85}

The available studies indicate that COVID-19 patients have hyperactive platelets that may contribute to the COVID-19 pathophysiology. Ongoing clinical trials are evaluating the effect of different platelet inhibitors in COVID-19 patients, such as NCT04368377, NCT04445623 and NCT04409834. We eagerly await the results of these trials.

Conclusion

Platelets play a role in the host immune response to viruses. They can engulf viral particles to reduce viremia. However, they can also enhance inflammation and tissue injury during respiratory viral infections. At present, we do not know if platelet inhibition will be beneficial in COVID-19 patients. More work is needed to determine the role of platelets in modulating the innate immune response to viral infections.