Keywords
platelets - hemostasis - inflammation - SARS-CoV-2 - COVID-19 - viral infections
Infectious diseases have significant impact globally, with high mortality and morbidity
rates reported each year by the World Health Organization.[1] Over the past few decades, new challenges associated with infectious diseases have
placed additional burdens on health care due to the emergence of antimicrobial resistance,[2] and viral pandemics including Ebola,[3] human immunodeficiency virus (HIV),[4] and most recently severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2),
which is causing the ongoing coronavirus disease 2019 (COVID-19) pandemic outbreak.[5]
Platelets are small anucleate cells derived from megakaryocytes, and are traditionally
known for their role in prevention of bleeding and minimizing vascular injury.[6] While vital for hemostasis, there has been an increasing awareness that platelets
also contribute to various human pathologies, including autoimmunity,[7] cancer,[8] and infectious diseases.[9] Thus, in addition to their key contribution to thrombus formation, there is increasing
consensus that platelets would play important roles in modulating immune and autoimmune
responses.[10]
[11]
The ability of platelets to participate in the immune response is in part due to their
ability to release a myriad of inflammatory and bioactive molecules stored within
their granules. These mediators are able to attract and modulate the activities of
circulating leukocytes, important in orchestrating localized immune responses to pathogens.[11] Platelets have also been found to elicit direct effector functions, so as to be
considered independent immune effector cells.[12] Indeed, megakaryocytes and platelets have been shown to express several immune-associated
molecules and receptors, including Fc receptors,[13] complement receptors,[14] chemokine receptors,[15]
[16] and an array of toll-like receptors (TLRs).[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25] The expression of these functional immune receptors raises the question whether
platelets can also engage viruses and contribute to antiviral immunity. In this short
narrative review, we explore how viruses engage circulating platelets and how they
contribute to viral pathology.
The Role of Platelets in Viral Immunity
The Role of Platelets in Viral Immunity
Viral immunity has traditionally focused on the roles of leukocytes, given their direct
involvement in viral spread and antiviral responses. Clinically, platelet hyperactivity
has been recognized as a hallmark of many viral infections, including dengue virus,[26] HIV,[27]
[28]
[29] influenza virus,[30] and SARS-CoV-2.[31] Given the prominent clinical presentations of platelet-driven events, along with
their emerging immune role, having a better understanding of the role of platelets
in viral infections may disclose and highlight novel therapeutic targets.
A key antiviral platelet response is to sequester viral particles, thus limiting viral
spread within the host environment. Evidence of such activity has been seen in HIV,
where platelets bind and endocytose HIV virions,[32]
[33]
[34]
[35] which is believed to help clearance of viral particles from circulation.[36] In addition to engaging viruses, platelets are also able to exert direct antiviral
properties. During platelet activation, α-granules are trafficked to the cell surface
and externalized, so releasing a wide spectrum of bioactive molecules, including platelet
factor 4 (PF4; also referred to as the chemokine CXCL4). As well as being an important
chemotactic agent for leukocytes, PF4 has direct antiviral activity, being found to
suppress HIV infection of T cells.[37]
[38]
[39] Interestingly, platelets may also help control infection through the secretion of
platelet antimicrobial peptides, such as PD1–PD4, which have been shown to have antiviral
activity against the vaccinia virus.[40] A recent study has also presented data demonstrating that platelets contain virus-specific
immunoglobulin G (IgG), which is able to potentially neutralize in vitro and in vivo
viral infection against human cytomegalovirus (HCMV) and influenza A virus.[41] Platelet-derived IgG localizes to α-granules,[42] suggesting that megakaryocytes are able to take up IgG, where they are stored in
α-granules for later secretion by mature platelets. Interestingly, IgG released from
platelets was found to be more efficient at neutralizing virus compared with equal
amounts of plasma IgG,[41] the biological significance of which is unclear.
Platelets are also able to orchestrate local immune responses to viral infection.
HCMV can be recognized by platelet TLR-2. This engagement leads to platelet degranulation,
leukocyte chemotaxis, and formation of platelet aggregates with neutrophils, monocytes,
B cells, T cells, and dendritic cells.[43] Through these platelet–leukocyte interactions, platelets present viral antigens
to leukocytes via major histocompatibility complex class I,[44] as well as providing co-stimulatory signals to antigen-presenting cells,[45] both of which can prime and mount an antiviral leukocyte response. Similar inflammatory
activities have been observed in dengue virus infection, whereby dengue-infected platelets
were able to induce monocyte activation.[46]
While platelets can exert a degree of antiviral immunity, viruses have evolved mechanisms
of evading platelet recognition. Viruses are able to engage with receptors at platelet
surfaces; for example, dengue virus and HIV both bind surface lectin receptors and
dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)
on platelets.[47] Such interactions lead to internalization of viral particles, where viruses such
has HIV, HCMV, and hepatitis C virus (HCV) can continue to replicate and generate
productive viruses within both megakaryocytes and platelets.[48]
[49]
[50] In addition to using platelets as a site of replication, some viruses use circulating
platelets as cellular carriers to evade immune detection, such as the influenza virus[51] and HIV,[52]
[53] essentially forming latent viral reservoirs within the circulation. Interestingly,
HCV is believed to utilize circulating platelets to transport itself to the liver,
where enhanced platelet–hepatocyte interactions prolong the time for potential viral
infection.[54]
Thrombocytopenia is a common feature among various viral infections, which is associated
with more severe diseases.[55] Viruses have developed several mechanisms to target and reduce platelet production
and/or integrity. A classic example can be seen with neuraminidase activity of influenza
virus, which reduces platelet life span by targeting them for rapid clearance in the
liver and spleen.[56] In addition to targeting platelets for destruction, neuraminidase activity also
alters megakaryocyte ploidy, morphology, and subsequent platelet size.[57] Human herpes viruses have also adopted similar mechanisms, and can interfere with
thrombopoietin activity, thus reducing megakaryocyte colony formation[58] and impairing megakaryocyte survival and differentiation.[59] Defective megakaryocyte differentiation can also be achieved by altering cytokine
expression in the bone marrow, which has been found in dengue virus infection.[60] By targeting these megakaryocytic developmental checkpoints, abnormal platelet activation,
mitochondrial dysfunction, reduced cellular integrity, and increased apoptosis are
often seen in patients infected with dengue,[61]
[62]
[63] encephalomyocarditis virus,[23] and HIV.[64]
[65]
In addition to impacting platelet integrity, viral infection can also affect platelet
function.[66] Coxsackievirus B virus (CVB) binds and enters platelets via the Coxsackie–Adeno
receptor.[67] While unable to replicate inside the platelet, CVB modulates activity and enhances
P-selectin release and phosphatidylserine exposure, which collectively promote platelet–leukocyte
interactions and ultimately leads to platelet destruction and thrombocytopenia,[67] driving viral pathology. While vaccinia virus is known to bind and enter platelets,[68] the significance of this interaction to disease is not completely understood. Early
studies reported reduced in vitro platelet aggregation, but increased serotonin release
in vaccinia-infected platelets,[68] which would suggest that platelet function may be suppressed. In contrast, in vivo
models found vaccinia virus infection led to fatal intravascular coagulation,[69] implicating an augmented platelet response. This disparity may highlight that the
vaccinia virus may impact endothelial function, which is known to be critical in regulating
in vivo platelet responses.[70]
[71]
Given the complex nature of the immune network, the existence of indirect effects
of viruses on platelets is unsurprising. Platelet hyperactivity in influenza infection
can be partially attributed to influenza's impact on monocyte cytokine release, which
then activates platelets.[72] Adaptive immune responses to HCV, HIV, HCMV, herpes viruses, and coronaviruses result
in the production of antibodies targeting viral glycoproteins to help neutralize and
suppress viral spread. These antiviral glycoprotein antibodies can, however, cross-react
with platelet integrins and trigger platelet autoantibody-induced thrombocytopenia
in several viral settings.[73] Viruses have also been found to infect the endothelium, with indirect effects on
platelet function. For example, dengue virus and hantaviruses infect endothelial cells,
promoting endothelial activation, endothelial–platelet interactions, and increasing
vascular permeability.[74]
[75] This disruption of vascular integrity is thought to contribute to the increased
platelet reactivity observed in virally infected patients and may represent one mechanism
of enhanced platelet clearance.
An additional consideration of chronic viral infection, like HIV, is that patients
require permanent therapeutic intervention to suppress viral replication. Some cohort
studies have found that certain antiretroviral drugs are associated with increased
risk of myocardial infarction,[76]
[77] which have subsequently been found to enhance platelet activation and aggregation.[78]
[79] These effects can be further enhanced by vascular endothelium, which is also impacted
by antiretroviral drugs in ways that increase platelet reactivity.[80]
[81] Such data demonstrate that both viral infection and therapeutic measures to control
infection can impact platelet reactivity.
The Platelet Response during SARS-COV-2 Infection
The Platelet Response during SARS-COV-2 Infection
Dual activation of inflammation and coagulation pathways, combined with an excessive
recruitment and activation of immune cells to sites of infection, is known as “immunothrombosis,”
a concept that was initially conceptualized by Engelmann and Massberg, in 2013, to
accurately define the crosstalk between hemostasis and the innate immune system.[82] Given that aberrant platelet activation has been documented with other viral infections,
researchers have begun to explore the potential contributory role of platelets to
SARS-CoV-2 infection.
The core pathology of COVID-19 is pulmonary, with epithelial cell infection by SARS-CoV-2
ultimately resulting in pulmonary leukocyte infiltration and an excessive inflammatory
response.[83] Clinical evidence supports this model, with several reports detailing signs of epithelial
and endothelial inflammation, leukocyte recruitment, and platelet activation in the
lung of COVID-19 patients.[84]
[85]
[86] Poorer prognoses in patients are shown to associate with abnormal coagulation parameters,
primarily D-dimer, fibrinogen, fibrin degradation product levels, reduced mitochondrial
depolarization, and phosphatidylserine exposure,[87]
[88]
[89] suggesting that thrombosis may be important to COVID-19 pathophysiology. Severe
pulmonary inflammation and obstructive immunothrombosis in the lung microvascular
network of COVID-19 patients, leading to pulmonary thrombosis/thromboembolism, underlie
multiple organ failure and mortality in patients with advanced stages of illness.[90]
[91]
[92]
Elevated plasma levels of proinflammatory cytokines such as interleukin (IL)-1α, IL-1β,
IL-6, IL-12, monocyte chemoattractant protein-1, interferon-γ, and tumor necrosis
factor-α have been found in patients with COVID-19.[93]
[94]
[95] While there is evidence of elevated proinflammatory cytokines, it is important to
note that there are also reports finding similar or lower levels of proinflammatory
cytokines when compared with patients with COVID-19-unrelated acute respiratory distress
syndrome or other cytokine release syndromes.[96]
[97]
[98] In addition, reports have also found elevated D-dimer concentrations in patients
with COVID-19,[99] which is consistent with the observed systemic inflammation and macrovascular thrombotic
complications seen in patients with SARS-CoV-2 infection,[100]
[101] and may therefore be linked to coagulation activation and diffuse macro- and microvascular
thrombosis.[102]
[103]
While SARS-CoV-2 messenger RNA (mRNA) can be detected in platelets isolated from patients
with COVID-19,[94]
[104]
[105]
[106] it is not clear whether SARS-CoV-2 is internalized by the platelets via receptor-mediated
endocytosis. Although it is widely accepted that SARS-CoV-2 infects host cells via
binding angiotensin-converting enzyme 2 (ACE2),[107] it is not known whether platelets express this protein. While some studies have
shown that neither ACE2 mRNA nor protein could be detected in platelets,[94]
[104] others have reported robust ACE2 expression in platelets, associated to direct platelet
activation by SARS-CoV-2 via spike/ACE2 interactions.[108]
[109] The reason for this disparity is unclear, but may stem from differences in washed
platelet preparation given that one study used sodium citrate-evacuated blood tubes,
while others used an acid/citrate/dextrose anticoagulant. It is also feasible that
genetic differences between cohort populations may account for differences in ACE2
expression or protein polymorphisms. These discordant results were clearly highlighted
in a recent review.[106] Interestingly, while Zaid et al demonstrated that platelets only associate with
SARS-CoV-2 RNA, they reported substantial alterations in the platelet transcriptome
and proteome profiles,[104] as well as platelet hyperreactivity.[94]
[104] The abilities of viruses including SARS-CoV-2 to associate and internalize with
platelets are listed in [Table 1].
Table 1
Associations and/or internalizations between platelets and different viruses
|
Virus
|
Platelet
|
References
|
|
Type
|
Nomenclature
|
Association
|
Internalization
|
|
DNA
|
Herpes simplex virus type 1 (HSV-1)
|
Yes
|
?
|
[108]
|
|
Human cytomegalovirus (HCMV)
|
Yes
|
?
|
[118]
|
|
Vaccinia virus (VACV or VV)
|
Yes
|
?
|
[43]
|
|
RNA
|
Human immunodeficiency virus (HIV)
|
Yes
|
Yes
|
[68]
|
|
Hepatitis C virus (HCV)
|
Yes
|
Yes
|
[32]
|
|
Dengue virus (DENV)
|
Yes
|
Yes
|
[119]
|
|
Influenza virus (flu virus)
|
Yes
|
Yes
|
[120]
|
|
Coxsackievirus B (CVB)
|
Yes
|
Yes
|
[121]
|
|
Encephalomyocarditis virus (EMCV)
|
Yes
|
Yes
|
[67]
|
|
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
|
Yes
|
?
|
[94]
|
These studies indicate that platelet activation contributes to COVID-19 pathophysiology.
Autopsy studies found evidence of extensive thrombosis in multiple organs,[84] suggesting enhanced platelet reactivity may be a driver of thrombosis in severe
COVID-19. Abnormal platelet morphology has also been reported in COVID-19 patients,
with evidence of large, hyperchromatic, and vacuolated platelets.[110] A recent study documented COVID-19 patients as having enhanced platelet hyperreactivity
relative to non-COVID-19 patients and controls subjects.[111] Greater levels of platelet–monocyte and platelet–granulocyte aggregates can be seen
in patients with COVID-19 pneumonia,[112] highlighting greater levels of systemic platelet activation. Further phenotypic
analysis revealed that resting platelets in COVID-19 patients had similar levels of
P-selectin expression as control platelets activated with collagen.[112] Taus et al also demonstrate that COVID-19 platelets contribute to the increased
fibrinogen, von Willebrand factor, and factor XII reported in patients, while facilitating
accelerated factor XII-dependent coagulation.[112] Moreover, platelets isolated from patients with severe COVID-19 were able to induce
ex vivo tissue factor expression in monocytes isolated from health controls,[88] indicating platelet crosstalk into other circulating cells. Interestingly, normal
platelet function is restored in patients who have recovered from SARS-CoV-2 infection,
which suggests that platelet hyperreactivity may be a direct consequence of SARS-CoV-2
infection.[111] Together, these data suggest that in COVID-19, platelets are primed to spread proinflammatory
and procoagulant activities within the systemic circulation.
[Fig. 1] summarizes the role that the platelet could have following SARS-CoV-2 infection.
Fig. 1 Hypothetical model for platelet interaction with SARS-CoV-2. SARS-CoV-2 potentially
depends on ACE2 receptor for viral entry through the endothelium and spread in the
host. Then various platelet receptors can mediate binding to viral particles; however,
SARS-CoV-2 binds to platelets probably via its potential receptor ACE2, and viral
hemagglutinin is cleaved by TMPRSS2 to activate internalization of the virus. Such
cleavage triggers platelet activation and downstream signaling events leading to cytokine
overproduction, platelet aggregation, and leukocyte–platelet aggregate formation.
The combination of the cytokine storm, platelet activation, microvesicle shedding,
and immunothrombotic events have deleterious consequences such as cellular damage,
acute lung injury, and thromboembolism (created with BioRender and Servier). ACE2,
angiotensin-converting enzyme 2; CLEC, C-type lectin-like receptor; CXCR, chemokine
receptor; DC-SIGN, dendritic cell-specific ICAM-grabbing nonintegrin; EV, extracellular
vesicles; FcγRIIa, low-affinity immunoglobulin gamma Fc region receptor II-a; MHC-1,
major histocompatibility complex 1; SARS-CoV-2, severe acute respiratory syndrome
coronavirus 2; TLR, toll-like receptor; TNFR, tumor necrosis factor receptor.
Conclusion
COVID-19 is a viral infection with variable clinical outcomes, determined by the amplitude
of immunothrombosis response and extent of tissue injury. While hyperinflammation
and the “cytokine storm” may be central to the most severe COVID-19 cases,[113] given the clinical spectrum of COVID-19, the absolute centrality of the “cytokine
storm” may not be as straightforward. It could be argued that the extent and importance
of increased cytokine release on pathology draws upon multiple factors including genetic,
host and viral phenotypic, and environmental.[114] Recently, studies using the bronchoalveolar lavage of COVID-19 patients in intensive
care, highlighted the presence of a ‘lipid storm’ but were unable to definitively
demonstrate whether platelets or other cells are the cause of this altered lipid profile.[115] Currently, available studies suggest that the COVID-19 coagulopathy comprises a
combination of localized pulmonary platelet consumption, low-grade disseminated intravascular
coagulation, and thrombotic microangiopathy.
Of particular interests are the various circulating inflammatory coagulation biomarkers
involved directly in clotting, with specific focus on fibrin/fibrinogen, D-dimers,
P-selectin, von Willebrand factor multimers, soluble thrombomodulin, and tissue factor,
which may amplify inflammation and hypercoagulability in patients with COVID-19. Central
to the activity of these biomarkers are their receptors and signaling pathways on
endothelial cells, platelets, monocytes, and erythrocytes. Altogether, these collective
observations raise the question as to whether the virus acts directly on the hemostatic
system or whether hemostatic activation is secondary to the upstream inflammatory
process.
Currently, literature remains ambivalent regarding ACE2 expression on platelets. It
would therefore be useful to explore whether SARS-CoV-2 directly binds platelets via
ACE2 or through alternative pathways. These studies may give better insight into underlying
pathways driving the “cytokine storm coagulation” that contributes to the multiple
organ dysfunction associated with severe COVID-19. While therapeutic intervention
targeting the cytokine storm in severe COVID-19 is gaining increasing attention,[116] the use of antiplatelet therapy also warrants further study. Aspirin administration
has been associated with a reduced risk of mechanical ventilation, intensive care
unit admission, and in-hospital mortality in 412 hospitalized COVID-19 patients.[117] A limitation to this study, however, is that it is a retrospective, observational
cohort study, which limits its strength as clinical evidence. This study does demonstrate
that further randomized controlled trials examining the efficacy of antiplatelet therapeutics
in treating patients with severe COVID-19 are of clinical value. These future trials
would be strengthened by complementary basic and translational studies dissecting
the role of platelets in COVID-19 pathophysiology.
Ultimately, a cross-disciplinary approach drawing upon the expertise of biomedical
and clinical communities is critical in developing a therapeutic arsenal to target
not only the cytokine storm but also the coagulopathy related to SARS-CoV-2 infection.
A deeper understanding of the contributions of platelets to viral immunity will not
only allow for better treatment of COVID-19, but also help to be more prepared to
manage future viral pandemics.
