key: cord-0885462-04oehpub
authors: Hottz, Eugenio D.; Bozza, Patrícia T.
title: Platelet‐leukocyte interactions in COVID‐19: Contributions to hypercoagulability, inflammation, and disease severity
date: 2022-05-02
journal: Res Pract Thromb Haemost
DOI: 10.1002/rth2.12709
sha: 3d86a3f93610054cc986650283016a98dceed50d
doc_id: 885462
cord_uid: 04oehpub

A State of the Art lecture titled “Platelet‐leukocyte interactions in COVID‐19: Contributions to hypercoagulability, inflammation and disease severity” was presented at the International Society for Thrombosis and Hemostasis (ISTH) congress in 2021. Severe coronavirus disease 2019 (COVID‐19) has been associated with a high incidence of coagulopathy and thromboembolic events that contributes to disease severity and poor outcomes. Therefore, understanding the mechanisms of COVID‐19‐associated hypercoagulability and thromboinflammation has gained great interest. Here, we review the mechanisms involved in platelet activation and platelet interactions with leukocytes during COVID‐19. We highlight recent evidence that platelet activation, platelet‐monocyte, and platelet‐neutrophil interactions in COVID‐19 support pathological thromboinflammation, including in driving tissue factor expression and NETosis, which have been associated with thromboembolic complication and poor outcomes in critically ill patients. The contributions of platelet‐leukocyte interactions to COVID‐19 immunoregulation, inflammation, and hypercoagulability, as well as their potential implications in disease severity and therapeutic strategies, will be discussed. Finally, we summarize relevant new data on this topic presented during the 2021 ISTH Congress.

When the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) completes its second anniversary, the coronavirus disease 2019 (COVID-19) pandemic will have already accumulated more than 300 million cases and 5 million deaths globally. 1 A state of hypercoagulability is a major pathophysiological mechanism and the main cause of mortality in severe COVID-19. [2] [3] [4] Individuals with severe COVID-19 syndrome frequently evolve with prothrombotic coagulation abnormalities, especially pulmonary embolism and deep vein thrombosis. 5, 6 Higher frequency of COVID-19associated thromboembolic events, cardiovascular complications, and death during postdischarge is also observed. 7, 8 This hypercoagulable state is associated with respiratory distress syndrome in SARS-CoV-2-infected patients. 9, 10 Postmortem pathological findings show extensive areas of platelet-fibrin microvascular thrombosis containing neutrophil and macrophage infiltration, NETosis, and endothelial inflammation ( Figure 1A ). [9] [10] [11] [12] [13] [14] Platelet-neutrophil complexes and NET-containing pulmonary and extrapulmonary microvascular thromboses have been presented as a main mechanism of multiorgan impairment in autopsy studies from COVID-19 fatalities. 9, 10, 12, 15 Pulmonary histopathological studies revealed these thromboinflammatory vascular occlusions to be almost 10 times more frequent in COVID-19 than in influenza pneumonia fatalities. 11, 12, 15 Aggravating an already complex situation, hypercoagulability and thromboinflammatory tissue damage have been reported despite prophylactic heparin anticoagulation, 10, 16, 17 and antiplatelet therapy with P2Y12 inhibitors fails to improve hypercoagulability and organ impairment, 18 highlighting the need of alternative antithrombotic strategies.

Platelet activation is a main feature of the COVID-19 hypercoagulable state. 19, 20 Platelet activation in severe COVID-19 patients is associated with markers of coagulation activation and inflammation 10, 19, 21 and with increased incidence of thromboembolic complications. 22 Platelets from severe COVID-19 patients are hyperresponsive to agonist stimulation and form aggregates with F I G U R E 1 Platelet activation and hypercoagulability in COVID-19. (A) COVID-19-associated microvascular thrombosis: thromboinflammatory vascular occlusions presenting neutrophil and macrophage infiltration with NET-containing platelet-fibrin thrombi are observed in multiple organs during severe COVID-19. (B) Mechanisms of platelet activation and platelet-endothelial cell aggregation: soluble mediators in COVID-19 plasma including cytokines, procoagulant antibodies, coagulation, and complement factors, as well as SARS-CoV-2 itself, may participate in platelet and endothelial cell activation. Activated endothelial cells promote platelet aggregation through von Willebrand factor. (C) Platelet-monocyte and platelet-neutrophil aggregates formation in COVID-19: platelets induce monocyte TF expression through P-selectin and integrin α IIb /β 3 signaling and neutrophil release of TF-containing NETs through mechanisms depending on Thrombin-PAR-1 and C5aR signaling monocytes, lymphocytes, and neutrophils. 10, 19, 20 Through the use of different strategies to inhibit platelet activation and plateletleukocyte aggregate formation, a role for platelets and plateletleukocyte interactions in fueling coagulation and inflammation disturbances in COVID-19 has been examined. In this review, we highlight mechanisms and pathophysiological roles of platelet activation and platelet-leukocyte interactions in hypercoagulability, inflammation, and severity during COVID-19.

Increased platelet activation in COVID-19 has been evidenced by alpha and dense granules release, integrin α IIb /β 3 activation, and platelet extracellular vesicle shedding. 10, 14, [19] [20] [21] [23] [24] [25] [26] Also, platelets from COVID-19 patients were more adhesive to fibrinogen and collagen and more responsive to PAR-1 and P2Y 12 agonists, leading to TXA 2 synthesis, aggregation, and cytokine secretion. 10, 14, 20, 23, 27 Platelet transcriptome analysis has revealed pathways involved in thromboinflammation, type I interferon-mediated antiviral response, and programmed cells death, which were associated with functional alterations in platelets from COVID-19 patients. 14, 20, 27, 28 These features of platelet activation and hyperresponsiveness were correlated to hypercoagulability, disease severity, respiratory distress syndrome, myocardial injury, and mortality in different cohorts of COVID-19 patients. 10, 19, 21, 29, 30 The mechanisms underlying platelet activation in severe COVID- 19 are not yet completely understood. Our group and others have shown that circulating factors present in the plasma of severe COVID-19 patients activate platelets ex vivo. 19, 31, 32 These circulating factors may include heightened thrombin generation, cytokine signaling, complement activation, and/or pro-coagulant antibodies ( Figure 1B) . 31, [33] [34] [35] [36] [37] [38] Platelets from healthy volunteers become activated when exposed to plasma from COVID-19 patients, 19, 32, 35, 39 and whole blood reconstituted in COVID-19 plasma displays platelet activation, platelet-leukocyte aggregate formation, and tissue factor (TF) expression, which are all inhibited by the interleukin-6 receptor antibody tocilizumab. 31 Anti-phospholipid antibodies are highly frequent in severe COVID-19 patients. Higher levels of anti-cardiolipin antibodies correlate with coagulation activation, NETosis, and respiratory distress in infected patients. 34 Immunoglobulins from patients have major thrombogenic effects, including triggering platelet activation, apoptosis, platelet-thrombi formation and NET extrusion in vitro, [34] [35] [36] 38 and driving intravascular NETosis and accelerated thrombosis in mice. 34 Aberrant glycosylation of anti-Spike antibodies and activation of Syk and PI3K/Akt signaling through FcγRIIa and C5aR are major signaling pathways in these processes. 33, 35, 36, [38] [39] [40] Downstream inhibition of FcγRIIa/ITAM signaling through Syk inhibitor or the Bruton tyrosine kinase inhibitor ibrutinib completely abrogate the thrombogenic effect of anti-S antibodies, highlighting possible pathways for therapeutical intervention. 36 These data highlight an interplay between innate and adaptive immune factors in triggering platelet activation and thromboinflammatory vascular occlusion in COVID-19.

Another thrombogenic mechanism of COVID-19 patients' plasma involves endothelium activation through C5a-C5aR1 signaling, inducing Weibel-Palade bodies extrusion and von Willebrand factor-dependent platelet aggregation ( Figure 1B ). 37 Similarly, the releasate from COVID-19 patients' platelets also activates a thromboinflammatory program in endothelial cells. 14 In vitro infection of platelets from healthy volunteers shows that platelets recognize and respond to SARS-CoV-2 becoming activated. 21, 28, 41, 42 Similar results have been observed in platelets exposed to recombinant Spike protein and Spike-containing pseudovirus. 21,41,43 SARS-CoV-2-induced platelet activation in vitro is inhibited by competition with recombinant ACE-2, suggesting platelet activation through the Spike receptor-binding domain. 41 However, whether platelets express or not ACE-2 has been a matter of controversy. 20 21, 27, 42, 43 Neutralizing antibodies against CD147, but not ACE-2, prevent platelet activation after exposure to SARS-CoV-2, Spike-pseudovirus, or recombinant Spike protein. 21 Similarly, Spike protein binds to CD42b and triggers Akt-protein kinase C pathway to induce platelet activation. 43 Of note, SARS-CoV-2 RNA has been detected in platelets from infected patients, 23 patients and from in vitro infection models. 21, 27, 28, 42 However, even though the subgenomic viral RNA is detected in infected platelets, no viral progeny is produced, indicating an abortive replication cycle. 21 

Increased platelet-leukocyte aggregates formation in COVID-19 patients has been reported among the main leukocyte subsets, including neutrophils, monocytes, and CD4+ and CD8+ T lymphocytes. 14, 20 Platelet-monocyte and platelet-neutrophil aggregate formation were especially higher in severe COVID-19 patients, and were associated with hyperinflammation and hypercoagulability. 10, 19, 32, 44 Importantly, circulating platelet-monocyte and plateletneutrophil aggregates in severe COVID-19 patients express high levels of TF, 19, 31, 33 the main trigger of intravascular coagulation and thrombosis ( Figure 1C ). 45 Increased platelet-monocyte aggregate formation has been shown in severe COVID-19 patients, but not in patients presenting mild COVID-19 syndrome or asymptomatic infections. 19 Increased TF expression was observed on monocytes that were tethered with platelets compared with monocytes alone in the same sample, whereas isolated platelets from severe COVID-19 patients triggered TF expression in monocytes from healthy volunteers. 19 Mechanistically, platelets from severe COVID-19 patients induced monocyte TF expression depending on P-selectin and integrin α IIb /β 3 signaling ( Figure 1C ). 19 Platelets activated by SARS-CoV-2 Spike protein also bind to monocytes through P-selectin and CD40-L, and signal proinflammatory monocyte activation with high IL-1β expression. 43 Similarly, results from our group show that platelets from COVID-19 patients or in vitro-infected platelets also induce a proinflammatory program in monocytes, which is induced by P-selectin and integrin α IIb /β 3 binding and is amplified by TF signaling. 46 Platelet activation and monocyte TF expression positively correlate with plasma levels of D-dimers, supporting a role in hypercoagulability. 19 In addition, increased platelet activation and monocyte TF expression at admission were predictive of patients' poor outcomes, including the requirement of mechanical ventilation and mortality. 19 These data highlight key roles of platelet-monocyte interaction in triggering pathological TF expression, contributing to hypercoagulability and inflammation in severe COVID-19 (Table 1) .

alongside platelet-fibrin thrombi in lungs, kidneys, and heart histological preparations from COVID-19 autopsies ( Figure 1A) . 9, 10 Increased NETosis is associated with hypercoagulability, respiratory distress syndrome, and Sequential Organ Failure Assessment score values during COVID-19; and NETosis at admission is predictive of venous thromboembolism and mortality. 9,10,32,33 Isolated neutrophils from COVID-19 patients show features of activation and spontaneously release NETs 9,33 that are decorated with TF. 33 Plasma from COVID-19 patients also activates control neutrophils and significantly induces NET extrusion ex vivo. 9, 32, 47 Similarly, anti-phospholipid antibodies isolated from COVID-19 patients also induce NETs release in neutrophils from healthy volunteers. 34 In another study, however, plasma from COVID-19 patients induced neutrophil TF expression without inducing NETosis, while platelet rich plasma (PRP) was required to induce the release of TF-containing NETs. 33 Neutrophil extrusion of TF-containing NETs in response to PRP from COVID-19 patients depended on Thrombin-PAR-1 and C5aR signaling ( Figure 1C ). 33 Collectively, these data highlight complex platelet-leukocyte interactions as key events for monocyte and neutrophil thromboinflammatory responses in severe COVID-19, including in driving TF expression and hypercoagulability (Table 1 ). 

Although platelet-neutrophil and platelet-monocyte aggregate formation are well documented, platelet-lymphocyte interactions have been far less explored. The ability of platelets to present antigens through HLA class I has been previously shown. 58, 59 Recently, platelet antigen presentation was associated with CD8+ T-cell suppression in sepsis. 58 On the other hand, platelet antigen presentation has been previously shown to activate CD8+ T-cell effector responses TA B L E 1 Platelet phenotypes and clinical correlates in COVID-19

Associated with hypercoagulability (D-dimer and fibrinogen), requirement of mechanical ventilation and mortality (19) Activation of ERK/p38/PLA 2 leading to TXA 2 synthesis and platelet hyperaggregability (20) Associated with inflammation, hypercoagulability, and disease severity (10)

Associated with critical illness and viremia (41) Activated platelets from COVID-19 patients increase factor XII formation in control plasma (65) Associated with ICU admission myocardial injury and mortality (29) Platelet hyperactivity is observed regardless of presenting ARDS

Increased shedding of platelet-derived factors and EVs

High levels of TXB 2 , sCD40L, and sCD62-P in plasma associated with increased risk of mortality and thrombosis (22) Platelet shedding of sCD62-P and HMGB-1-containing EVs associated with inflammation (CRP), hypercoagulability (D-dimer), respiratory distress (PaO 2 /FiO 2 ), and disease severity (21) High levels of sCD62-P in plasma predictive of mechanical ventilation and mortality (30) Platelets from patients secrete higher levels of IL-1β and sCD40-L. PF4 and serotonin are reduced in platelets and augmented in plasma from patients (23) High levels of sCD62-P, sGPVI, and PF4 in plasma (42) Increased platelet apoptosis Increased in ICU-admitted patients and associated with thrombocytopenia, hypercoagulability, SOFA score, thrombosis, and mortality. IgG fraction from COVID-19 serum activates platelets through FcγRIIA (38) Morphological features of platelet activation, cell shrinking, and cell death. MLKL phosphorylation and caspase-3 cleavage were observed in platelets that were positive to Spike protein both in patients and after SARS-CoV-2 infection in vitro

Increased platelet-neutrophil aggregates and NETosis

NETosis in blood, airways, and postmortem lung histopathological analysis were associated with respiratory distress (PaO2/FiO2), requirement of mechanical ventilation, SOFA score, and mortality

Neutrophil activation and NETosis are observed in blood and in lung, kidneys, and heart histopathological analysis. Associated with hypercoagulability and respiratory distress. PRP from COVID-19 patients induce NETosis in control neutrophils (10) .

Platelet-neutrophil aggregate formation was associated with disease severity and with the inflammatory markers (CRP and IL-6)

Release of NETs decorated with TF. PRP from COVID-19 patients induced TF-positive NETs in control neutrophils through PAR1 and C5aR signaling

Increased TF expression in platelets and platelet-neutrophil aggregates. Plasma from COVID-19 patients induces a similar phenotype in control blood, which is prevented by aspirin, P2Y12 inhibitors, or IL-6R blocking (31) NETosis but not platelet activation was associated with ICU admission, requirement of mechanical ventilation, and VTE

Increased platelet-monocyte aggregates Platelets induce TF expression in monocytes through CD62P and integrin α IIb /β 3 signaling. Monocyte TF expression was associated with hypercoagulability (D-dimer and fibrinogen), requirement of mechanical ventilation, and mortality.

Associated with disease severity and with inflammatory markers (CRP and IL-6)

Increased TF expression in platelets and platelet-leukocyte aggregates. Plasma from COVID-19 patients induces a similar phenotype in control blood, which is prevented by aspirin, P2Y12 inhibitors, or IL-6R blocking (31) Platelets form aggregates especially with CD16+ inflammatory monocytes. Platelet-monocyte interactions reciprocally activate monocytes and platelets, inducing the secretion of inflammatory mediators. Platelet adhesion is a primary signaling mechanism inducing mediator secretion and TF expression, whereas TF activity amplifies inflammation by inducing TNFα and IL-1β through PAR1 and 2 signaling (46) Abbreviations: ADP, adenosine diphosphate; ARDS, acute respiratory distress syndrome; C5aR, complement factor 5a receptor; CRP, C-reactive protein; EVs, extracellular vesicles; HMGB-1, high mobility group box 1; ICU, intensive care unit; NET, neutrophil extracellular traps; PAR, proteaseactivated receptor; PAR1, protease-activated receptor 1; PF-4, platelet factor 4; PLA 2 , phospholipase A 2 ; PRP, platelet-rich plasma; sCD40L, soluble CD40 ligand; sCD62P, soluble P-selectin; sGPVI, soluble glycoprotein VI; SOFA, Sequential Organ Failure Assessment; TF, tissue factor; TRAP, thrombin receptor-activating peptide; TXA 2 , thromboxane A 2 ; VTE, venous thromboembolism in malaria. 59 Therefore, platelet-lymphocyte interaction is potentially involved in T-cell features with major implications to COVID-19 pathophysiology, including immunosuppression through T-cell exhaustion or inflammatory amplification and tissue damage through cytotoxic activity.

As the knowledge on COVID-19 pathophysiology advances, Postdischarge thromboprophylaxis may be beneficial. 7 However, new studies are still necessary to investigate the participation of platelets and platelet-leukocyte aggregate formation in the persisting inflammation and thromboembolic risk of COVID-19 survivors.

The work of the authors is supported by CNPq, CAPES, FAPERJ, FAPEMIG, and INOVA/FIOCRUZ.

The authors declare that they have no conflicts of interest relevant to the content of this manuscript. P.T.B. has received fees as lecturer for GSK.

Both authors wrote and edited the manuscript.

Eugenio D. Hottz https://orcid.org/0000-0002-2201-1742

Patrícia T. Bozza https://orcid.org/0000-0001-8349-9529

Eugenio D. Hottz @EugenioHottz

COVID-19) dashboard | WHO coronavirus (COVID-19) dashboard with vaccination data

Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia

Incidence of thrombotic complications in critically ill ICU patients with COVID-19

Incidence of venous thromboembolism in hospitalized patients with COVID-19

COVID-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: JACC state-of-the-art review

COVID-19-associated coagulopathy and disseminated intravascular coagulation

Postdischarge thromboembolic outcomes and mortality of hospitalized patients with COVID-19: the CORE-19 registry

Long-term cardiovascular outcomes of COVID-19

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Immunothrombotic dysregulation in COVID-19 pneumonia is associated with respiratory failure and coagulopathy

A Descriptive and quantitative immunohistochemical study demonstrating a spectrum of platelet recruitment patterns across pulmonary infections including COVID-19

Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19

Vascular disease and thrombosis in SARS-CoV-2-infected rhesus macaques

Platelets amplify endotheliopathy in COVID-19

Vascular neutrophilic inflammation and immunothrombosis distinguish severe COVID-19 from influenza pneumonia

In vitro hypercoagulability and ongoing in vivo activation of coagulation and fibrinolysis in COVID-19 patients on anticoagulation

Effect of intermediatedose vs standard-dose prophylactic anticoagulation on thrombotic events, extracorporeal membrane oxygenation treatment, or mortality among patients with COVID-19 admitted to the intensive care unit: the INSPIRATION randomized clinical trial

Effect of P2Y12 inhibitors on survival free of organ support among non-critically Ill hospitalized patients with COVID-19: a randomized clinical trial

Platelet activation and platelet-monocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Platelet gene expression and function in patients with COVID-19

Unconventional CD147-dependent platelet activation elicited by SARS-CoV-2 in COVID-19

Platelet and vascular biomarkers associate with thrombosis and death in coronavirus disease

Platelets can associate with SARS-CoV-2 RNA and are hyperactivated in COVID-19

Platelet reactivity to thrombin differs between patients with COVID-19 and those with ARDS unrelated to COVID-19

Lipid bodies are reservoirs of cyclooxygenase-2 and sites of prostaglandin-E2 synthesis in colon cancer cells

Expansion of myeloid derived suppressor cells contributes to platelet activation by L-arginine deprivation during SARS-CoV-2 infection

Platelets contribute to disease severity in COVID-19

SARS-CoV-2 initiates programmed cell death in platelets

Over time relationship between platelet reactivity, myocardial injury and mortality in patients with SARS-CoV-2-associated respiratory failure

Platelet activation in critically ill COVID-19 patients

Platelet and endothelial activation as potential mechanisms behind the thrombotic complications of COVID-19 patients

Association of neutrophil activation, more than platelet activation, with thrombotic complications in coronavirus disease 2019

Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19

Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia

Aberrant glycosylation of anti-SARS-CoV-2 spike IgG is a prothrombotic stimulus for platelets

C5a and C5aR1 are key drivers of microvascular platelet aggregation in clinical entities spanning from aHUS to COVID-19

Antibody-induced procoagulant platelets in severe COVID-19 infection

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19

Antibody-mediated procoagulant platelet formation in COVID-19 is AKT dependent

SARS-CoV-2 binds platelet ACE2 to enhance thrombosis in COVID-19

SARS-CoV-2 interacts with platelets and megakaryocytes via ACE2-independent mechanism

Platelets mediate inflammatory monocyte activation by SARS-CoV-2 spike protein

Neutrophil-platelet and monocyte-platelet aggregates in COVID-19 patients

Tissue factor: an essential mediator of hemostasis and trigger of thrombosis

Platelet-monocyte interaction amplifies thromboinflammation through tissue factor signaling in COVID-19

Neutrophil extracellular traps in COVID-19

Systemic platelet exhaustion in critically-ill covid-19 patients -ISTH Congress abstracts

Altered platelet proteome and platelet-leukocyte interactions in patients with COVID-19 -ISTH Congress abstracts

Impact of COVID-19 on platelet activity and the platelet releasate -ISTH Congress abstracts

COVID-19 infection is associated with antibody-mediated procoagulant platelets: data from an observational study -ISTH Congress abstracts

Lipid-binding antiphospholipid antibodies trigger autoimmune signaling in severe COVID-19 -ISTH Congress abstracts

Platelet activation via PI3K/AKT signaling pathway in COVID-19 -ISTH Congress abstracts

cAMP prevents antibody induced thrombus formation in COVID-19 -ISTH Congress abstracts

Blockade of COVID-19 antiphospholipid antibody-induced thrombosis by a specific inhibitor of the TF initiation complex -ISTH Congress abstracts

SARS-CoV2 spike protein can activate platelets through integrin alpha IIb beta 3 -ISTH Congress abstracts

Platelet factor 4 (PF4) enhances in vitro neutrophil extracellular traps (NET) capture of coronaviruses: clinical and therapeutic implications -ISTH Congress abstracts

Platelet MHC class I mediates CD8+ T cell suppression during sepsis

Platelets present antigen in the context of MHC class I

1-year outcomes in hospital survivors with COVID-19: a longitudinal cohort study

Long-COVID": a cross-sectional study of persisting symptoms, biomarker and imaging abnormalities following hospitalisation for COVID-19

Postdischarge thrombosis and hemorrhage in patients with COVID-19

Prolonged elevation of Ddimer levels in convalescent COVID-19 patients is independent of the acute phase response

Persistent endotheliopathy in the pathogenesis of long COVID syndrome

Platelets promote thromboinflammation in SARS-CoV-2 pneumonia

Plateletleukocyte interactions in COVID-19: Contributions to hypercoagulability, inflammation, and disease severity