key: cord-1014304-mr3fyqgr
authors: Petito, E; Falcinelli, E; Paliani, U; Cesari, E; Vaudo, G; Sebastiano, M; Cerotto, V; Guglielmini, G; Gori, F; Malvestiti, M; Becattini, C; Paciullo, F; De Robertis, E; Bury, L; Lazzarini, T; Gresele, P
title: Neutrophil more than platelet activation associates with thrombotic complications in COVID-19 patients
date: 2020-12-06
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa756
sha: 725e1eff5f2ca617c8d8df3507b96c8b9a8d6d55
doc_id: 1014304
cord_uid: mr3fyqgr

BACKGROUND: SARS-CoV-2 infection is associated with hypercoagulability which predisposes to venous thromboembolism (VTE). We analyzed platelet and neutrophil activation in COVID-19 patients and their association with VTE. METHODS: Hospitalized COVID-19 patients and age- and sex-matched healthy controls were studied. Platelet and leukocyte activation, neutrophil extracellular traps (NETs), and matrix metalloproteinase-9 (MMP-9), a neutrophil-released enzyme, were measured. Four patients were re-studied after recovery. The activating effect of COVID-19 plasma on control platelets and leukocytes and the inhibiting activity of common antithrombotic agents on it were studied. RESULTS: 36 COVID-19 patients and 31 healthy controls were studied; 8/36 COVID-19 patients (22.2%) developed VTE. Platelets and neutrophils were activated in COVID-19 patients. NET, but not platelet activation, biomarkers correlated with disease severity and were associated with thrombosis. Plasmatic MMP-9 was significantly increased in COVID-19 patients. Platelet and neutrophil activation markers, but less so NETs, normalized after recovery. In vitro, plasma from COVID-19 patients triggered platelet and neutrophil activation and NET formation, the latter blocked by therapeutic dose low-molecular weight heparin, but not by aspirin or dypiridamole. CONCLUSIONS: Platelet and neutrophil activation are key features of COVID-19 patients. NET biomarkers may help to predict clinical worsening and VTE, and may guide LMWH-treatment intensity.

Matrix metalloproteinase-9 (MMP-9) SARS-CoV-2

A c c e p t e d M a n u s c r i p t 5 Background COVID-19 is a rapidly spreading viral pneumonia caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) declared pandemic by the World Health Organization on March 2020 [1] .

Besides respiratory system involvement, SARS-CoV-2 infection induces severe blood clotting alterations with an unbalance towards hypercoagulability [2, 3] predisposing to venous thromboembolism (VTE), arterial and microvascular thrombosis as a result of the strong systemic inflammatory reaction [4] . Indeed, abnormal coagulation parameters and high concentrations of proinflammatory cytokines are associated with disease severity, poor prognosis and with the incidence of VTE in COVID-19 patients [2, 5, 6] .

Thrombocytopenia and elevated blood neutrophil counts have also been associated with disease severity and mortality in hospitalized COVID-19 patients, suggesting that these blood cells are deeply involved in the evolution of the infection [7, 8] .

While several studies have assessed coagulation and platelet count alterations in SARS-CoV-2 infection, only few focused on platelet and leukocyte activation, and on their relationship with VTE incidence [9] [10] [11] [12] [13] [14] .

Viral infections initiate a systemic inflammatory response stimulating the blood clotting system, a process called thromboinflammation in which platelet activation and plateletneutrophil interactions play a crucial role [15] . Indeed, platelets act as blood sentinels quickly detecting invading pathogens through pattern recognition receptors (e.g., TLRs), get activated and form platelet-neutrophil complexes [15, 16] . The interaction with activated platelets triggers the release by neutrophils of neutrophil-extracellular traps (NETs), 3-dimensional extracellular lattices composed by DNA, citrullinated histones, granule-derived enzymes and cytoplasmic proteins generated to limit infections but which may, when deranged, propagate inflammation and thrombosis [17] .

A c c e p t e d M a n u s c r i p t 6 Activated neutrophils release also microparticles (PMN-MPs) which have been linked to thrombosis in vasculitis, inflammatory bowel disease and acute coronary syndromes [18, 19] . PMN-MPs complex with NETs during sepsis enhancing thrombin generation, further highlighting the role of neutrophils in thrombosis [18] . Circulating PMN-MPs have not been studied so far in COVID-19 patients. NET formation in plasma and sera of COVID-19 patients, as well as their presence in autoptic lung specimens, have instead recently been reported, but no data on their correlation with VTE were provided [9, 11, 20] .

Neutrophils contain in their tertiary granules and release upon activation matrix metalloproteinase-9 (MMP-9) which binds to NETs and induces endothelial dysfunction [21] . MMP-9 is also involved in neutrophil migration in lungs in response to viral infections [22] . Given that a strong neutrophil infiltration has been shown in the pulmonary microthrombi and in the bronchial tissue of COVID-19 patients [23] , it is conceivable that MMP-9 may contribute to lung damage and/or thrombosis in COVID-19 patients. MMP-9 exists as a heterodimer with neutrophil gelatinase-associated lipocalin (MMP-9/NGAL) which prevents its inactivation, increasing MMP-9 proteolytic activity [24] .

Increased plasmatic levels of MMP-9/NGAL have been reported in patients with pulmonary embolism, especially in the more severe cases [25] . Based on these considerations and on the pivotal role of MMPs in acute respiratory distress syndrome/acute lung injury, these enzymes have been recently postulated as a possible therapeutic target to prevent severe lung injury in COVID- 19 [26] , but MMP-9 levels in COVID-19 patients have not been measured so far.

Here we analyzed platelet and neutrophil activation and MMP-9 and MMP-9/NGAL levels in COVID-19 patients, focusing on their role in thrombosis. Moreover, we evaluated the effect of common antithrombotic agents in preventing COVID-19 patient plasma-triggered platelet and neutrophil activation and NET formation. 

Patients. Hospitalized COVID-19 patients were enrolled in a multicenter study carried out in the Umbria region approved by the local Ethics Committees (CEAS Umbria n. 3656/20

and University of Perugia Bioethics Committee n. 2020-36346) and each study participants or their legally authorized representative gave written informed consent in accordance with the Declaration of Helsinki. Patients were studied on average 3.4±0.6 days (95%CI 2.1-4.8) after the last positive nasopharyngeal swab. Thirty-one age-and sex-matched healthy volunteers who had not taken agents influencing platelet or leukocyte function in the previous seven days were also enrolled. Exclusion criteria for healthy controls were history of systemic autoimmune disease, active infection, allergic disorders and pregnancy. Four patients were restudied 2-3 months after hospital discharge made after 2 negative nasopharyngeal swabs (mean 72.7 days, 95% CI 32.8-112.6). Demographic, hemodynamic and prognostic variables, including PaO2/FiO2 ratio and sequential organ failure assessment (SOFA) score, were recorded.

Platelet and neutrophil activation markers. Citrated venous blood was collected and platelet P-selectin expression, platelet-leukocyte complexes, platelet-and neutrophil- 

Clinical and demographic characteristics of enrolled COVID-19 patients and healthy subjects are reported in Table 1 

We evaluated as platelet activation markers platelet P-selectin, soluble P-selectin and circulating PMPs [28] . A significant increase in platelet surface P-selectin expression was found in COVID-19 patients compared to healthy controls ( Figure 1A ). Soluble P-selectin (s-Psel) and PMPs were also significantly enhanced ( Figure 1B indicating that the degree of platelet activation is related to COVID-19 severity, confirming previous results [12] . [18, 19, 29] , were significantly increased in COVID-19 patients compared to healthy controls ( Figure 2A ). It is known that neutrophil activation leads to the release of NETs which play a crucial role in thromboinflammation [17] , we thus measured two highly specific markers of NET formation in plasma. Myeloperoxidase (MPO)-DNA complexes ( Figure 2B ) and citrullinated histone H3 (citH3) ( Figure 2C (TIMP-1), a selective natural MMP-9 inhibitor binding to the MMP-9 active site in a 1:1 stoichiometric ratio, was also elevated in COVID-19 patients, but the ratio MMP-9/TIMP-1, which expresses residual MMP-9 proteolytic activity, was still significantly higher than in healthy controls ( Figure 4C, D) , showing that MMP-9 activity is enhanced in patient plasma.

No significant differences were found between COVID-19 patients admitted into ICU and non-ICU wards, between patients requiring or not mechanical ventilation and between patients who developed thrombotic events or not for any of the MMP-9-related biomarkers (data not shown).

Moreover, no differences were observed between COVID-19 patients treated with prophylactic dose LMWH (n=25), and COVID-19 patients who did not receive it (n=8) (data not shown).

In patients who had recovered from COVID-19 a normalization of platelet P-selectin expression, PMPs, platelet-leukocyte and platelet-neutrophil complexes and MMP-9 was [2] [3] [4] . Platelets play a connecting role between haemostasis and immunity getting activated by invading pathogens, crosstalking with neutrophils and contributing to thromboinflammation [15] which triggers VTE and microvascular thrombosis [30, 31] . Activated neutrophils release NETs and microparticles (PMN-MPs), which induce endothelial damage and platelet activation [32, 33] , playing an important role in VTE [17] .

Here we confirm that circulating platelets and neutrophils are strongly activated in COVID-19 patients [9, 10] , but we also show for the first time that NET formation, more than platelet activation, is associated with thrombotic events. We also report for the first time that COVID-19 patients have significantly increased plasma levels and activity of MMP-9. Finally, we show that plasma from COVID-19 patients induces platelet and neutrophil activation and NET formation which are prevented by high but not low concentrations of LMWH.

Differently from previous studies [10, 12] , our COVID-19 patients were mainly mild but still we found strong platelet and neutrophil activation, showing that the SARS-CoV-2 infection itself rather than the multiorgan dysfunction associated with severe forms triggers these blood cell function modifications [34] . On the other hand, platelet and neutrophil activation markers correlated with COVID-19 severity, suggesting that the On the other hand, we show for the first time that NET formation more than platelet activation is associated with thrombosis in COVID-19 patients. In fact, while for all platelet activation parameters no significant difference was found between patients with thrombotic events and those without, NET biomarkers were associated with thrombosis, showing moderate accuracy in discriminating patients developing VTE from those not. Interestingly, 7 of the 8 COVID-19 patients with thrombotic events were males, confirming the higher VTE risk in males [36], and male COVID-19 patients had significant higher NET, but not platelet activation, biomarkers compared to females further supporting the conclusion that neutrophils associate with thrombotic complications.

Although platelets play a role in NET formation [15, 16] , several other pathways are also involved [37,38,39], including neutrophil activation by pro-inflammatory cytokines which are greatly increased in SARS-CoV-2 infection [35] , and this may explain the stronger association of NETs respect to platelet activation with VTE. This central pathogenic role of NETs in COVID-19-associated VTE is in agreement with findings in cancer-associated thrombosis, another thromboinflammatory condition [15, 40] , and in animal models of viral infections [41] . No significant differences were found in PMN-MPs between COVID-19 patients who developed thrombosis or not, suggesting that it is specifically NETosis rather than simply neutrophil activation to be implicated in COVID-19-associated thrombosis.

We also show for the first time that MMP-9 and the MMP-9/NGAL heterodimer as well as the MMP-9/TIMP1 ratio, an index of MMP-9 proteolytic activity, are significantly We then tested whether some common antithrombotic agents (aspirin, dipyridamole and LMWH), prevent platelet and neutrophil activation induced by COVID-19 plasma. In one observational study in severe SARS-CoV-2 infection, dipyridamole-treated patients improved platelet counts and D-dimer levels [11] . We show that while aspirin and dipyridamole did not attenuate the platelet-and neutrophil-triggering activity of COVID-19 plasma, LMWH prevented NET formation in a dose-dependent manner, being significantly effective at a concentration corresponding to the therapeutic dose. The fact that the patients who developed VTE in our case series were under LMWH does not contradict its effectiveness in preventing NET formation concentration-dependently.

Indeed, previous studies have shown a high frequency of VTE in LMWH-treated patients [46] but the incidence in untreated patients is unknown as no randomized studies comparing standard or intensified prophylactic-dose LMWH with placebo have been performed so far. Moreover, it has been suggested that LMWH dose should be increased from prophylactic towards therapeutic in high risk patients, like those hospitalized in ICU or with high D-dimer [47, 48] . The dose-dependent ability of heparin to dismantle the NET scaffold has previously been reported [49] . Therefore, our results support the importance of LMWH prophylaxis for COVID-19 patients [3] , and suggest that NET biomarkers may guide the switching to potentiated prophylactic dose or, in high-risk patients, to therapeutic dose to prevent neutrophil contribution to VTE [48] .

In conclusion, we show that a strong in vivo platelet and neutrophil activation occurs in hospitalized COVID-19 patients, including in not severe cases, and that NETs most probably significantly contribute to the development of thrombosis. Our findings corroborate the idea that NET formation represents an important therapeutic target to reduce the thrombotic complications of COVID-19 [9, 23] .

A c c e p t e d M a n u s c r i p t 17 We recognize that a limitation of our study is the small number of COVID-19 patients who developed thrombotic events: further studies in larger patient cohorts are warranted to validate the predictive value of NET biomarkers for COVID-19-related thrombosis.

However, while our manuscript was under review, a paper associating NET levels with the risk of developing thrombotic events has been published, supporting our conclusions [50] .

Moreover, our findings concur to the hypothesis that MMP-9 may contribute to neutrophil infiltration in lungs [9, 23] and that it might represent a potential therapeutic target for the prevention of lung damage in COVID-19 patients [26].

In summary, our results suggest that plasmatic NET biomarkers may be used for prognostication of COVID-19 severity and VTE risk and encourage the investigation of novel NET-targeting approaches for the prevention of thrombotic complications [9, 20, 23] . 

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The authors declare no conflict of interest, except for Dr. Becattini Cecilia that reports personal fees from Bayer HealthCare, from Daiichi Sankyo, from Bristol Myers Squibb, and from Pfizer, outside the submitted work.

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 20