key: cord-0951300-ibbi72g2
authors: Maugeri, Norma; De Lorenzo, Rebecca; Clementi, Nicola; Antonia Diotti, Roberta; Criscuolo, Elena; Godino, Cosmo; Tresoldi, Cristina; Angels for COVID‐BioB Study Group, Bio; Bonini, Chiara; Clementi, Massimo; Mancini, Nicasio; Ciceri, Fabio; Rovere‐Querini, Patrizia; Manfredi, Angelo A.
title: Unconventional CD147‐dependent platelet activation elicited by SARS‐CoV‐2 in COVID‐19
date: 2021-11-16
journal: J Thromb Haemost
DOI: 10.1111/jth.15575
sha: 36735e3022d807d8edfc5890311272f49844eef2
doc_id: 951300
cord_uid: ibbi72g2

BACKGROUND: Platelet activation and thrombotic events characterizes COVID‐19. OBJECTIVES: To characterize platelet activation and determine if SARS‐CoV‐2 induces platelet activation. PATIENTS/METHODS: We investigated platelet activation in 119 COVID‐19 patients at admission in a university hospital in Milan, Italy, between March 18 and May 5, 2020. Sixty‐nine subjects (36 healthy donors, 26 patients with coronary artery disease, coronary artery disease, and seven patients with sepsis) served as controls. RESULTS: COVID‐19 patients had activated platelets, as assessed by the expression and distribution of HMGB1 and von Willebrand factor, and by the accumulation of platelet‐derived (plt) extracellular vesicles (EVs) and HMGB1(+) plt‐EVs in the plasma. P‐selectin upregulation was not detectable on the platelet surface in a fraction of patients (55%) and the concentration of soluble P‐selectin in the plasma was conversely increased. The plasma concentration of HMGB1(+) plt‐EVs of patients at hospital admission remained in a multivariate analysis an independent predictor of the clinical outcome, as assessed using a 6‐point ordinal scale (from 1 = discharged to 6 = death). Platelets interacting in vitro with SARS‐CoV‐2 underwent activation, which was replicated using SARS‐CoV‐2 pseudo‐viral particles and purified recombinant SARS‐CoV‐2 spike protein S1 subunits. Human platelets express CD147, a putative coreceptor for SARS‐CoV‐2, and Spike‐dependent platelet activation, aggregation and granule release, release of soluble P‐selectin and HMGB1(+) plt‐EVs abated in the presence of anti‐CD147 antibodies. CONCLUSIONS: Hence, an early and intense platelet activation, which is reproduced by stimulating platelets in vitro with SARS‐CoV‐2, characterizes COVID‐19 and could contribute to the inflammatory and hemostatic manifestations of the disease.

Endothelial activation in various organs with the recruitment of inflammatory cells and activation of the coagulation system with venous thrombosis, pulmonary embolism and microvascular angiopathy/thrombosis are hallmarks of COVID-19. These events contribute to organ dysfunction in critically ill patients with SARS-CoV-2 1-3 and thrombocytopenia. The main function of platelets is indeed hemostatic. They patrol the vasculature and guard its integrity, undergoing activation in response to damaged endothelia. Moreover, platelets express surface receptors that directly interact with microbes 4 and contribute to the host response at various levels, via the release of microbicidal agents from granules and via the recruitment of cellular and humoral innate immunity.

Megakaryocytes and platelets are a preferential target of selected microbes, flaviviruses in particular, 5 and thrombocytopenia is common in acute viral infections.

A decrease in platelet counts, more frequent in patients with worse clinical outcomes, has been described in COVID-19. 6, 7 However, platelet counts per se may not be sufficiently informative in an ongoing infection, as thrombocytopenia may reflect a decrease in platelet production in the bone marrow, an increase in peripheral destruction and clearance of platelets or a combination of the two events. Conversely, platelet activation could result in the generation of bioactive extracellular vesicles (EVs) that reach distant sites through the circulatory system and perpetuate damage and inflammation in various organs, including the lung. [8] [9] [10] Recently, platelet activation in patients with COVID-19 has been independently described in various cohorts. [11] [12] [13] [14] [15] Differential gene expression profile of platelets of COVID-19 patients was associated with enhanced ability to aggregate and to form aggregates with leukocytes 11 and platelet activation was prominent in patients with severe disease. 12, 14 Additionally, a major platelet response to SARS-CoV-2 has recently been described, which included massive release of granule content, generation of microparticles, and eventual death by apoptosis or necroptosis. 16 ACE2, the only primary receptor identified so far, appears to be dispensable for the interaction between platelets and SARS-CoV-2. [16] [17] [18] Here, we have identified platelet-derived extracellular vesicles expressing the DAMP, HMGB1 as surrogate markers of platelet activation that predict the outcome of COVID-19, and the CD147 receptor as a critical player in the activation of host platelets following interaction with SARS-CoV-2. Characteristics of the cohort have been described. 1 All patients with COVID-19 with a positive SARS-CoV-2 real-time RT-PCR from a nasal and/or throat swab and for which we were able to timely obtain and process blood were studied. Data collected from chart review and patient interview were entered in a dedicated electronic case record form. Twenty-six patients with coronary artery disease (CAD), seven patients with severe sepsis, and 36 healthy donors served as controls (Table 1 ).

Monoclonal antibodies (mAbs) against CD61 (SZ21), CD62P (Thromb-6), irrelevant IgG isotype control, Flow-Count Fluorospheres, and Thrombofix were obtained from Beckman Coulter (Italy). mAbs against HMGB1 (clone 3E8) was from Biolegend (Italy). PGE1, thrombin receptor agonist peptide-6 (TRAP-6), and thrombin were obtained from Sigma (Italy 

• Activation of platelets in COVID-19 is heterogeneous.

• Platelets challenged with SARS-CoV-2 undergo activation, dependent on the CD147 receptor.

• Activated platelets release soluble P-selectin and HMGB1 + extracellular vesicles.

• Early accumulation of platelet HMGB1 + extracellular vesicles predicts worse clinical outcomes.

Venous blood were immediately fixed, stored at 4°C and platelet activation markers (P-selectin, HMGB1, and von Willebrand factor [VWF] expression) were analyzed on a daily aligned Navios flow cytometer (Beckman Coulter, Milan, Italy). 9, 10, 19 Quantification of platelet-derived EVs (plt-EVs) and assessment of HMGB1 expression were performed in platelet-free plasma, as previously described. 9, 10 The gating strategy is depicted in Figure 1A , H.

Vero E6 cells were cultured in Dulbecco's modified Eagle medium supplemented with nonessential amino acids, penicillin/ streptomycin, Hepes buffer, and 10% (vol/vol) fetal bovine serum (FBS). A clinical isolate of SARS-CoV-2 (hCoV-19/Italy/UniSR1/2020; GISAID accession n. EPI_ISL_413489) was obtained and propagated in Vero E6 cells.

Venous blood was obtained from healthy volunteers who had not received any pharmacological treatment in the previous 10 days. Venous blood was drawn through a 19-gauge butterfly needle. After discarding the first 3-5 ml, blood was carefully collected in tubes containing Na 2 EDTA. Samples were centrifuged at 150g, 10 min at 20°C, to obtain platelet-rich plasma COPD, n (%) 6 (5.0) 2 (28. Note: *p < 0.05, **p < 0.01, ***p < 0.001. Each control group (sepsis, HD, and CAD) was compared with the COVID-19 cohort. ‡Calculated as the time from hospital admission to death, discharge, or time of statistical analysis for patients still hospitalized.

ARDS, acute respiratory distress syndrome; BMI, body mass index; CAD, coronary artery disease; CKD, chronic kidney disease; COPD, chronic obstructive pulmonary disease; CRP, C-reactive protein; DM, diabetes mellitus; HD, healthy donor; HTN, arterial hypertension; ICU, intensive care unit; IQR, interquartile range; ; LDH, lactate dehydrogenase; LMWH, low-molecular weight heparin; PaO 2 /FiO 2 , arterial oxygen partial pressure/ fractional inspired oxygen.

(PRP) to be used to isolate platelets as described. 9 

Platelets were stimulated with TRAP-6 (25 µM) or thrombin (0.5 U/ ml) as positive controls. were analyzed. Platelets and platelet-derived extracellular vesicles (plt-EVs) were identified in the blood within the CD61 + population (threshold of acquisition) by their side scatter (y axis) and forward scatter (x axis) characteristics (A, gating strategy). Plt-EVs were identified within the CD61+ population in platelet-free plasma (H, gating strategy). Representative dot plots of a healthy donor and a patient with COVID-19 are shown A and H). Panels B-D depict the fraction of platelets expressing P-selectin, expressing HMGB1 and the fraction of HMGB1 + plt-EVs respectively. P-sel low and P-sel high platelets were identified as those with a percentage of activated platelets within or higher than the confidential range observed in healthy donors. High or low P-selectin expression is independent of the fraction of HMGB1 + platelets (F) or HMGB1 + plt-EVs (G). In contrast, the plasma concentration of soluble P-selectin was significantly higher in patients with P-sel low platelets (I) and correlated with platelet P-selectin surface expression (J). The concentration of plt-EVs and of HMGB1 + plt-EVs were both significantly higher in patients with COVID-19 than in healthy volunteers or patients with CAD (K, L) and was independent of P-selectin expression (M). Symbols depict individual observations in each subject and whiskies median, minimum, and maximum values. Statistical differences were determined by Kruskal-Wallis test followed by Dunn's multiple comparison test. and resuspended in complete medium supplemented with 2% FBS. 

Platelets were isolated and stimulated as described previously and 

Platelet aggregation and dense granule release assays were performed by Lumi-aggregometry (Chrono-Log, Mascia Brunelli, Milan). 22 PRP samples were challenged with recombinant SARS-CoV-2 Spike protein S1 subunit (Val16-Gln690) or with the Spike S1 subunit Arg319-Phe541 fragment at a final concentration of 30 ng/µl each. When indicated, challenge with the previous stimuli or with the platelet agonist, TRAP-6 was carried out in the presence of an irrelevant antibody, of mAbs against CD147 or of mAbs against ACE2 (Table S1 ). Samples were run in duplicate, with a total volume of 20 µl.

The nucleic acid extraction, cDNA synthesis, and detection of SARS-CoV-2 subgenomic mRNA were performed by adapting described protocols. 23 Briefly, 24 h after virus inoculation, platelets were col- Experiments were performed using platelets derived from three to five independent donors in duplicate. Platelets derived from the same donors not exposed to SARS-CoV-2 served as a negative control. 

Blood was retrieved from 119 patients with COVID-19 admitted at the emergency department of a university hospital in Milan, Italy, between March 18 and May 5, 2020. To assess markers of F I G U R E 2 HMGB1 + plt-EVs concentration is associated with COVID-19 severity and predicts clinical outcomes. The concentration of HMGB1 + plt-EVs correlates with the concentration of C-reactive protein (CRP; A), of D-dimer (B), with hypoxia as reflected by the arterial oxygen partial pressure/fractional inspired oxygen (PaO 2 /FiO 2 , C) and with the concentration of lactate dehydrogenase (LDH, D) while being significantly higher in patients who will develop acute respiratory distress syndrome (ARDS) (E) and who will experience more severe disease, as reflected by the maximum score achieved using the World Health Organization ordinal scale as a surrogate for clinical deterioration (F). Symbols depict individual observations in each subject. Statistical differences were determined as described in the Methods.

platelet activation, samples were immediately fixed and analyzed. Figure 1B) and of the prototype endogenous inflammatory signal, HMGB1

( Figure 1C ), markers that reflect platelet activation regardless of the original stimulus (Table S2) . After activation, platelets generate and release EVs (Table S2) .

Plt-EVs accumulated in the plasma of patients with COVID-19.

Plt-EVs and the fraction of plt-EVs expressing HMGB1, which play a direct role in pulmonary microvascular activation and inflammation, 9, 10 were significantly more concentrated in the blood of COVID-19 patients than in the blood of control subjects ( Figure 1D , M). Therefore, three independent markers, which are associated with platelet activation in patients with sepsis ( Figure 1B-D) , revealed platelet activation in the blood of COVID-19 patients.

Of interest, P-selectin expression was bimodal and 53 (45%) patients had a level of P-selectin on activated platelets similar to that of controls (Figure 1 , E) regardless of the fact that they quantitatively expressed high levels of HMGB1 and had high levels of plt-EVs expressing HMGB1 ( Figure 1E,G,M) . This feature could be specific for COVID-19 because activated platelets expressing low levels of P-selectin were not detectable in patients with severe sepsis ( Figure 1E ), a condition in which different pathways are involved in platelet activation.

Defective release of the content of α granules, where P-selectin is stored in platelets at rest, or accelerated shedding of P-selectin could contribute. The first event is unlikely, as other α granule components such as VWF were depleted in platelets of COVID-19 patients regardless of the extent of P-selectin membrane expression (Table 2 ). In support of the second mechanism, various proteases cleave P-selectin, which is for example released as a soluble molecule from activated platelets. 27, 28 In agreement, the concentration of soluble P-selectin was significantly higher in the plasma of patients with activated platelets expressing low levels of P-selectin ( Figure 1I ,J). Figure 2F ) along with age, comorbidities, extent of hypoxia and concentrations of CRP, LDH, and D-dimer at hospital admission (Table 3) . When adjusted for the previously mentioned predictors, the concentration of HMGB1 + plt-EVs survived as independent predictor of the clinical outcome on multivariate analysis (p < 0.001, Table 3 and Figure 2F ).

(SARS-CoV-2) or TRAP-6, as reflected by platelet HMGB1 expression, the concentration of released HMGB1 + plt-EVs, the VWF platelet content, the P-selectin expression, and the concentration of released soluble P-selectin in the supernatant (A). Representative morphological electron microscopies of platelet challenged with TRAP-6, with SARS-CoV-2 or with SARS-CoV-2 614G pseudovirus particles (B). Platelet aggregation after challenge with TRAP-6, recombinant Spike S1 proteins Val16-Glu 690, and Arg 319-Phe 541 (C). Time course (x axis, minutes) of platelet response as reflected by HMGB1 expression, concentration of released HMGB1 + plt-EVs, and platelet P-selectin expression after challenge with TRAP-6, with recombinant Spike S1 proteins Val16-Glu690 and Arg319-Phe541 or with the culture supernatant of cells transfected with the empty expression vector as negative control (D). *p < 0.0001, significantly different from resting platelets. Statistical differences were determined as described in the Methods.

We combined SARS-CoV-2 and human platelets (10 6 /µl) for an hour at 37°C. Platelets challenged with SARS-CoV-2 underwent activation, as demonstrated by HMGB1 expression, generation of HMGB1 + plt-EVs, depletion of platelet VWF ( Figure 3A ). Electron microscopy confirmed the activation of platelets, which acquired an approximately spherical shape and lost α and dense granules. This was associated with the fusion of the open canalicular and dense tubular systems, the centralization of the organelles and the peripheral arrangement of the mitochondria ( Figure 3B ). Platelet activation was replicated using pseudoviral particles (PP) expressing SARS-CoV-2 Spike protein ( Figure 3B ). Platelets were activated to a similar extent by Spike expressing pseudovirions, by the full-length Spike S1 subunit protein (Val16-Gln690) or by the S1 fragment containing the receptor-binding domain (Arg319-Phe541). Recombinant S1 proteins also effectively induced platelet aggregation and release reaction, with a lag-time of approximatively 5 min (5.1 ± 0.8 min for Spike S1 Val16-Glu690 and 4.3 ± 1.0 min for Spike S1 Arg319-Phe541) ( Figure 3C and Figure 4B ).

The time course of HMGB1 expression and of HMGB1 + Plt-EVs release indicates that full platelet response occurs after 60 min ( Figure 3D ). The kinetics of P-selectin expression is different because it increases in the first phase of the incubation to subsequently abate ( Figure 3D ), a pattern that might be compatible with eventual shedding of the molecule. In support, platelets challenged with purified SARS-COV-2 expressed relatively low amounts of P-selectin on the plasma membrane after 60 min, but soluble Pselectin accumulated at that time in the extracellular environment ( Figure 3A) .

ACE2, the best characterized receptor for SARS-CoV-2, is expressed in a fraction of human platelets only (2.7 ± 1.5% in platelets at rest, 3.3 ± 0.6% in activated platelets, Figure 4A ). CD147, a SARS-CoV-2 putative coreceptor, 26 is constitutively expressed on a large fraction of platelets from healthy donors (60 ± 18.5% in platelets at rest, 71.3 ± 18.1% in activated platelets, Figure 4A ). Platelet activation induced by SARS-CoV-2 PPs or Spike S1 recombinant proteins, assessed by measuring platelet expression of HMGB1, release of EVs and depletion of VWF, aggregation and release reaction abated in the presence of antibodies blocking the CD147 receptor, while antibodies that block ACE2 were ineffective (Figure 4 ,B, C).

The effect was specific, since CD147 blockade did not influence TRAP-6-elicited platelet activation (Table S1 ). The fraction of platelets expressing P-selectin in this system did not reflect the extent of activation in response to viral stimuli, despite the effective depletion of the alpha granules reflected by the VWF content. This discrepancy was probably due to the release of the soluble molecule into the environment. In agreement, the concentration of soluble P selectin in the supernatant of Spike-stimulated platelets was increased ( Figure 4C ). This event was significantly inhibited by blocking the CD147 receptor ( Figure 4C ).

The interaction between the SARS-CoV-2 protein Spike and platelets suggests the possibility that the virus infects them, even if we did not detect virus-like particles within platelets exposed to virus at analysis by electron microscopy ( Figure 3B ) Figure 5E ).

F I G U R E 4 SARS-CoV-2 activates platelets via CD147. Human platelets surface expression of ACE2 and CD147 at rest or after challenge with TRAP-6 or a SARS-CoV-2 clinical isolate (SARS-CoV-2). Histograms represent the % of platelets resting or stimulated with TRAP-6 or with SARS-CoV-2 expressing ACE2 or CD147 (mean ± SD) (A). ATP aggregation and release from dense granules monitored by lumiaggregometry in platelet-rich plasma after challenge with TRAP-6, Spike S1 Val16-Glu690, or Arg319-Phe541 recombinant proteins in the presence of an irrelevant mAbs (no inhibitors), of anti-CD147 or of anti-ACE2 Abs. Representative traces and quantification of maximal aggregation and ATP release are reported (B). Activation of platelets, as reflected by platelet HMGB1 expression, the concentration of released HMGB1 + plt-EVs, the VWF platelet content, the P-selectin expression and by the concentration of released soluble P-selectin were assessed after challenge with SARS-CoV-2 614D pseudoviral particles (PP), SARS-CoV-2 614G PP, Spike S1 Val16-Glu690, or Spike S1 Arg319-Phe541 recombinant proteins for 1 hr in the presence of irrelevant Abs (irr ab), of anti-CD147 or anti-ACE2 Abs. # p < 0.001, significantly different from platelets stimulated in the presence of irrelevant Abs or of anti-ACE2 Abs, determined by Kruskal-Wallis test followed by Dunn's multiple comparison test.

Platelets are guardians of vascular integrity. They vastly outnumber leukocytes in the blood and constantly patrol the vasculature for the integrity of the endothelial layer and the presence of infectious agents.

Platelets express receptors specialized in microbe recognition. 29 The best characterized receptor for SARS-CoV-2 is the ACE2, which is required for the infection of most nucleated cells.

However, the extent of the expression and the physiological relevance of ACE2 in human platelets are controversial. 11, 17, 18, 32 CD147, a receptor constitutively expressed on a large fraction of human platelets, has been described as a coreceptor involved in SARS-CoV-2 infection of epithelial cells in vitro and in vivo. 26 However, other groups failed to identify a direct role of CD147 in Spike-mediated infection of epithelial cells, 33, 34 suggesting that it may act more as an attachment cofactor than as a bona fide receptor needed for virus entry. 35 Our results indicate that platelet preparations that have been exposed to SARS-CoV-2 contain only traces of the virus, which has not actively replicated, suggesting that the receptor is not sufficient for platelet infection.

In support, a limited presence of SARS-CoV-2 in platelets of patients with COVID-19 has been reported by others 11 The role of platelets in intravascular immunity includes, on meeting a microbe in the circulation, the deployment of mechanisms to prevent its dissemination, including its containment within thrombi.

The process, referred to as immunothrombosis, depends on the ability of platelets to trigger the generation of neutrophil extracellular traps (NETs), 29 via presentation of their own HMGB1. 19, 40 Strongly supporting the role of immunothrombosis in COVID-19, the presence of thrombi containing NETs associated with platelets and fibrin in the pulmonary, renal, and cardiac microcirculation of patients with COVID-19 has been reported. [41] [42] [43] [44] Microthrombosis was related to the extent of neutrophil and platelet activation and interaction pattern in the blood, and to both ARDS and systemic hypercoagulability. [41] [42] [43] [44] Respiratory virions. 48 Therefore, the blood and possibly the pulmonary microcirculation could represent a privileged meeting point for the virus and platelets.

Platelets undergo early activation in the context of other infections in which the viral load in the blood is limited, such as human influenza. In these patients, platelet activation has been associated with lung involvement. 49 In the case of human influenza, Koupenova et al. have shown influenza virus engulfment by platelets, which results in the activation of neutrophils via a pathway that involves the complement cascade. 50 We did not find phagocytosis of SARS-CoV-2, possibly highlighting the different systems used by platelets to recognize the two viruses. In both cases, platelets undergo substantial activation and a role for complement in COVID-19 has been convincingly proposed. 41, 51 Further studies are warranted to verify whether platelet and neutrophil activation and complement involvement are causally related in COVID-19.

In this study, we observe widespread activation of platelets in patients with COVID-19, which correlates with activation of the coagulation cascade, with exposure of HMGB1 on the platelet membrane and with generation of HMGB1 + plt-EVs, which are bioactive elements that activate endothelial cells, favor the generation of NETs and cause lung inflammation. 9, 10 Our data support the rising COVID-19 model depicting platelet activation and coagulopathy as interconnected events via the recruitment of inflammatory leukocytes. Of importance, there is a significant association between the extent of platelet activation at admission and disease outcomes (this report and previous work [11] [12] [13] [14] [15] 31 ), suggesting that platelet activation might be a target for molecular intervention in COVID-19. 

Early predictors of clinical outcomes of COVID-19 outbreak in

Coagulation abnormalities and thrombosis in patients with COVID-19

COVID and coagulation: bleeding and thrombotic manifestations of SARS-CoV2 infection

Platelets endocytose viral particles and are activated via TLR (toll-like receptor) signaling

Dengue virus binding and replication by platelets

subjects with COVID-19

Haematological characteristics and risk factors in the classification and prognosis evaluation of COVID-19: a retrospective cohort study

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

Platelet microparticles sustain autophagy-associated activation of neutrophils in systemic sclerosis

Platelet phagocytosis via PSGL1 and accumulation of microparticles in systemic sclerosis

Platelet gene expression and function in COVID-19 patients

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

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

Platelet activation and plasma levels of furin are associated with prognosis of patients with coronary artery disease and COVID-19

Platelets promote thromboinflammation in SARS-CoV-2 pneumonia

SARS-CoV-2 initiates programmed cell death in platelets

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

Is there a role for the ACE2 receptor in SARS-CoV-2 interactions with platelets?

Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps

Clearance of circulating activated platelets in polycythemia vera and essential thrombocythemia

Oxidative stress elicits platelet/leukocyte inflammatory interactions via HMGB1: a candidate for microvessel injury in sytemic sclerosis

Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function

SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication

Acute respiratory distress syndrome: the Berlin definition

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial

CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells

Circulating soluble P-selectin must dimerize to promote inflammation and coagulation in mice

P-selectin and platelet clearance

Platelet TLR4 activates neutrophil extracellular traps to ensnare bacteria in septic blood

Endotheliopathy in COVID-19-associated coagulopathy: evidence from a single-centre, crosssectional study

Clinical features and outcome of COVID-19 pneumonia in haemodialysis patients

COVID-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome

Human basigin (CD147) does not directly interact with SARS-CoV-2 spike glycoprotein

No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor

Role of host factors in SARS-CoV-2 entry

Search for SARS-CoV-2 RNA in platelets from COVID-19 patients

Basigin (CD147), a multifunctional transmembrane glycoprotein with various binding partners

Expression of EMMPRIN (CD147) on circulating platelets in vivo

Extracellular cyclophilin A activates platelets via EMMPRIN (CD147) and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice

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

Vascular occlusion by neutrophil extracellular traps in COVID-19

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

Patients with COVID-19: in the dark-NETs of neutrophils

Virological assessment of hospitalized patients with COVID-2019

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19

High frequency of SARS-CoV-2 RNAemia and association with severe disease

SARS-CoV-2 Viremia is associated with COVID-19 severity and predicts clinical outcomes

In vivo platelet activation in critically ill patients with primary 2009 influenza A(H1N1)

The role of platelets in mediating a response to human influenza infection

Neutrophil extracellular traps (NETs) as markers of disease severity in COVID-19

The authors have declared that no conflict of interest exists.