key: cord-0989440-1rl7vxaa
authors: Lazzaroni, Maria Grazia; Piantoni, Silvia; Masneri, Stefania; Garrafa, Emirena; Martini, Giuliana; Tincani, Angela; Andreoli, Laura; Franceschini, Franco
title: Coagulation dysfunction in COVID-19: The interplay between inflammation, viral infection and the coagulation system
date: 2020-08-24
journal: Blood Rev
DOI: 10.1016/j.blre.2020.100745
sha: 3664945e2a51e70f94f32edab8563adfaff4ca41
doc_id: 989440
cord_uid: 1rl7vxaa

COVID-19 is a new pandemic, caused by Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-Cov2) infection and characterized by a broad spectrum of clinical manifestations. Inflammation and the innate immune system have been recently recognized as pivotal players in the most severe forms, characterized by significantly elevated levels of pro-inflammatory cytokines. In this setting, several studies have also reported the presence of abnormalities in coagulation parameters and platelets count, possibly identifying a subgroup of patients with poor prognosis. Some reports of full-blown thromboembolic events are emerging. Among the possible mechanisms underlying coagulation dysfunction, the so-called cytokine storm seems to play a pivotal role. Other candidate factors include virus-specific mechanisms, related to the virus interaction with renin angiotensin system (RAS) and the fibrinolytic pathway, but also comorbidities affecting these patients. Coagulation dysfunction is therefore a candidate risk factor for adverse outcomes in COVID-19 and should be carefully addressed in clinical practice.

with WMD -48x10 9 /L (95% CI -57 to -39 x10 9 /L). The prevalence of thrombocytopenia was reported in 4 of the 9 studies (1427 patients) and widely ranged from 7% to 41.7%.

At the same time, thrombocytosis was also recorded in the moderately severe cases with prolonged hospitalization [13] It was reported that in 94 patients with COVID-19 the levels of D-dimer and FDP were significantly higher than in 40 healthy controls during the same period and were also higher in severe forms as compared to milder forms [7] . Elevated levels of D-dimer at the time of admission were also identified as risk factor for mortality in a retrospective cohort of 191 Chinese patients [14] . Higher levels of D-dimer also emerged as one of the risk factors for ARDS and death in a series of 201 patients with COVID-19 pneumonia in China [8] . At the time of admission, 23.3% of them had D-dimer above the reference, while activated partial thromboplastin time (APTT) and PT were increased in 9.7% and 2.1% respectively, and platelets levels were reduced in 18.8%.

Noteworthy, elevated D-dimer levels have also been described and related to worst outcomes in patients with other acute lung diseases, such as community acquired pneumonia [15] , acute lung injury [16] and ARDS [17] .

Altogether, these findings indicate that elevated levels of D-dimer and FDP, prolonged PT and APTT could mark severe forms and possibly later stages of COVID-19 infection, characterized by poor prognosis.

On the contrary, the majority of patients, possibly those with milder forms or in earlier stages of the disease seems to have normal platelet levels and coagulation parameters; a minority can also show reactive thrombocytosis, possibly related to the acute inflammatory status, and reduced PT and APTT [18] . Therefore, coagulation dysfunction not only could reflect the severity of the disease among different patients, but in the same patient could vary according to the stage of the disease, suggesting a careful monitoring of these parameters during hospitalization [19] .

The prognostic role of coagulation parameters was also assessed in a clinical series with 449 COVID-19 patients, by demonstrating the role of heparin use >7 days during hospitalization in reducing the 28-days mortality [20] . This outcome was positively associated with D-dimer, PT and age, and negatively correlated with platelet count in the multivariate analysis.

The greatest benefit in terms of highest 28-days survival among the 99 heparin users was demonstrated for those with D-dimer levels >6 fold of upper limit of normal and "sepsis induced coagulopathy" (SIC) score ≥4. SIC is a novel definition provided by ISTH to define patients in earlier phases of sepsis associated DIC, who could experience the highest benefit from anticoagulant treatment.

To date, no experimental investigations on the coagulation cascade are available apart from data coming from clinical practice. Considering the similarities between this new pandemic and SARS infection, an upregulation of genes associated with the coagulation pathway could be hypothesized. In fact, the overexpression of procoagulant genes such as those of factors II, III and X, has been demonstrated in an in vitro model containing SARS-infected peripheral blood mononuclear cells [21] An impairment in fibrinolysis also seems to play a central role in COVID-19. A 'fibrinolytic shutdown' was demonstrated to be associated with thrombotic events in a clinical series of 44 patients, as revealed by thromboelastography measurements [22] . Moreover, another study taking advantage of thromboelastometry and considering 40 patients admitted to ICU, was not able to find any sign of secondary hyperfibrinolysis [23] .

As early as coagulation abnormalities were reported in many Chinese cohorts from Wuhan, clinical reports of thromboembolic events became increasingly reported.

Initially, some Chinese Authors have suggested an increased rate of thrombotic and thromboembolic events in these patients [19] , supported by their clinical practice and by similarities with the SARS infection, in which the rate of thrombotic events was estimated to be 20.5% for deep venous thrombosis (DVT) and 11.4% for pulmonary embolism (PE) in a report from Singapore [24] . One study also reported 5 SARS patients who developed large artery ischaemic strokes, but 3 of them were treated with high-doses Intravenous Immunoglobulins (IVIg), that could have enhanced the pro-coagulant status [25] .

One of the first studies from Wuhan, in which 1008 patients with COVID-19 were hospitalized between January and February 2020, retrospectively identified 25 patients who also underwent to computed tomography pulmonary angiography (CTPA) on the basis of clinical suspicion [26] . Among them, 10 had acute pulmonary embolism (APE), that was dominantly located in small branches of the pulmonary artery.

Interestingly, all 25 patients had increased D-dimer levels, but the 10 with APE had significantly higher levels as compared to the 15 without APE.

Subsequently, a large multicentre retrospective study showed an incidence of thrombotic complications in ICU patients around 30%, reinforcing the recommendations to consider an appropriate thrombotic prophylaxis in all COVID-19 ICU patients [27] . At the same time, in a single-centre retrospective study, the incidence of venous thromboembolism of 25% was reported by the same group of patients [28] .

Importantly, different studies reported the necessity of changing the usual schedule of thrombo-prophylaxis (including dose and duration), because of the excess of risk in COVID-19 cohort as compared to other patients with acute lung injury [29, 30] .

Moreover, from a clinical point of view, different studies have underlined the importance to consider demographic characteristics and comorbidities of each patient, to correctly estimate the thrombotic risk of COVID-19 patients. The role of concomitant risk factors should also be considered.

Elderly (70 years) patients are those prone to develop the most complicated infections as confirmed in a recent meta-analysis collecting the results of 147 studies on 20662 Chinese patients in which the mean patient age was 49 years and 53% of patients were male [31] . A significant prevalence of cardiovascular comorbidities was also described in a large Chinese cohort, including systemic hypertension (21%), diabetes mellitus (12%), cardiovascular (9%) and cerebrovascular diseases (6%) [31] . In another large report of 1099 J o u r n a l P r e -p r o o f Journal Pre-proof patients with confirmed COVID-19, systemic arterial hypertension and diabetes mellitus were recorded in 23.7% and 16.2% respectively [32] . In a report from the Italian Institute of Health 68% of 3032 patients who died for COVID-19 had hypertension, 30% diabetes, 28% ischemic heart disease and 11% obesity [33] .

Interestingly, acute cardiac injury determined by elevated high-sensitivity troponin levels is frequently reported in critically ill patients and is strongly associated with mortality. Different potential mechanisms for acute effects on the cardiovascular system during the infection have been proposed, including a direct infection of myocardial cells causing myocarditis, but also side effects of anti-viral treatments [34, 35] .

Cardiovascular diseases, such as hypertension, diabetes and obesity are often associated with Angiotensin/Angiotensin I-converting enzyme 2 (ACE2) dysregulation, which aggravates the imbalance caused by the infection, suggesting a role of these comorbidities as risk factors for a poor prognosis in COVID-19 [36] . Additionally, in the setting of ICU admission that concern critically ill patients, respiratory failure and prolonged mechanical ventilation further increase the risk of thrombotic events [37] . It is important to consider that patients with comorbidities take a concomitant variety of drugs. Alarm has emerged for ACE inhibitors and angiotensin-receptor blockers (ARBs), which are commonly used for hypertension. In some reports it was reported an increase of risk for SARS-CoV-2 infection because of the potential increasing of expression of the entry receptor of the virus (ACE2) [38] . On the contrary, other studies demonstrated that ARBs' treatment might reduce lung injury modulating the angiotensin type (AT) 1 receptors [39] . Globally, the latest data support the continued use of these drugs during the pandemic.  Specific-virus mechanisms.

During SARS-CoV2 infection elevated levels of different pro-inflammatory cytokines induced by the innate immunity activation have been described and the so called "cytokine release syndrome" seems to be responsible of the most severe manifestations of the disease [3] . Among these cytokines, a prominent role of interleukin-6 (IL-6) has emerged and is currently addressed in clinical trials with anti-IL-6 agents to treat severe forms [40] .

In vitro, most pro-inflammatory cytokines have been demonstrated to activate the coagulation system; in vivo, high levels of tumor necrosis factor (TNF), IL-6 and IL-1 are detectable in patients with acute inflammatory conditions (such as sepsis) together with a hyper-coagulable status, sometimes evolving in DIC [41] . Results from clinical trials in sepsis with drugs targeting these pathways, showed that IL-6 rather than TNF seems to be the most important mediator for cytokine-induced coagulation activation. The role of IL-1 is uncertain, as its levels in sepsis become detectable significantly after the appearance of coagulation abnormalities [41] .

IL-6 could also stimulate megakaryopoiesis [42] and has a prominent role in the induction of tissue factor (TF) expression in inflamed tissues. Therefore, TF expression could be enhanced in the lungs of patients affected by COVID-19, both via direct exposure due to tissue damage and inflammation, and via increased expression induced by IL-6 [43] .

IL-6 promotes also the synthesis of other coagulation factors such as fibrinogen and factor VIII [44, 45] and, acting on endothelial cells, induces vascular permeability by stimulating VEGF secretion [46] . It was reported that soluble IL-6 receptor (IL-6R) is abnormally elevated in the plasma of COVID-19 patients as consequence of enhanced cleavage from the cells surface during the infection. Afterwards, the circulating complexes of soluble IL-6 and IL-6R can activate directly most cells, including endothelial cells [47] . It's possible that the infection-related continuous coagulopathy may contribute also to the thrombocytopenia and, at the same time, the cytokine storm could stimulate the proliferation of megakaryocytes with consequent thrombocytosis [13] . The opposite platelets' alterations could reflect different phases of this disease, but their underlying specific mechanisms have to be elucidated [9] .

SARS-CoV-2 belongs to beta-coronavirus genus, enveloped and positive-stranded RNA viruses. Spike surface glycoprotein of the virus engages ACE2, which is an integral membrane receptor expressed in many J o u r n a l P r e -p r o o f Journal Pre-proof cells, including the lung, heart, kidney and intestine. The physiological function of ACE2 is to counterregulate ACE activity by reducing angiotensin II availability [48] . The virus-mediated engagement of ACE2 decreases its expression and activates the renin-angiotensin system (RAS), promoting platelet adhesion and aggregation [49] (Figure 1 ). Moreover, RAS is known to exert substantial control over the fibrinolytic system, with strong evidences that it represents a mediator in the association between reduced fibrinolytic activity and ischemic clinical events in patients with systemic hypertension [50] . In fact, pharmacologic interventions that reduce the activity of angiotensin II also have positive effects on fibrinolytic balance and frequency of cardiovascular events.

In particular, high levels of Plasminogen Activator Inhibitor-1 (PAI-1), the principal inhibitor of fibrinolysis interfering with tPA and urokinase, have been related with increased risk of thromboembolic events [50] .

Interestingly, ACE2 was recognized to play a crucial role as receptor also for in vivo infection by SARS-CoV [51] . In a report including 16 patients with SARS-CoV infection, the blood levels of PAI-1 were significantly higher as compared to 20 healthy controls, but also to 19 patients with infectious pneumonia of other etiology [52] . These findings were consistent with another study, in which the PAI-1 mRNA and protein levels in the human hepatoma cell line Huh7 infected with SARS-coronavirus were much higher as compared to infection with human Coronavirus 229E (associated with the common cold), as evidenced by transcriptome experiments, qRT-PCR and ELISA [53] , thereby explaining a possible direct effect of infection on the production of anti-coagulant factors. Furthermore, by infecting Serpine1-knockout mice, some Authors showed that the urokinase pathway had a significant effect also on lung pathology in SARS-CoV [54] . Plasminogen also contributes to inflammation caused by influenza virus through fibrinolysis [55] , confirming the strictly connection between virus-induced lung injuries and abnormalities in the coagulation system.

During SARS-CoV pneumonia the expression of platelet derived vitronectin (VN), a mediator of platelet adhesion, was also dramatically increased but it was uncertain whether it originated from increased expression by the liver or from lung damage [56] .

It was demonstrated that SARS-CoV2 can infect primary endothelial cells in vitro [57] and there is some evidence of the infection of endothelial cells in severe cases of COVID-19 [58] . The replication within endothelial cells induces cell death inducing the activation of pro-coagulant reactions [59] .

Another possible virus-specific mechanism could be related to the induction of autoimmunity, that was also described in SARS patients, as a consequence of molecular mimicry mechanisms [60] .

Moreover, antiphospholipid antibodies (aPL), recognized as risk factors for arterial and venous thrombosis, have been associated with different viral infections, such as parvovirus B19, several herpes viruses, hepatitis viruses and human immunodeficiency virus. Whether these autoantibodies contribute to clinically important thrombotic events during infections remains controversial [60] . Some evidences that lupus anticoagulant activity (LA) and anti-beta2 glycoprotein I antibodies were induced after immunization with viral peptides were found [61] . On the other hand, in some reports aPL were transient and not predictive of thrombotic complications [62] . Interestingly, post-infectious aPL were reported to have distinct immunochemical J o u r n a l P r e -p r o o f characteristics; for example, anti-cardiolipin antibodies after viral infections were described to be non-beta2 glycoprotein I-dependent, differently from those detectable in autoimmune diseases [63] .

Furthermore, in some patients the presence of infection-related aPL was also associated with the clinical manifestations of antiphospholipid syndrome, especially when other risk factors for thrombosis (such as inherited thrombophilia) were present [64] .

A first case-report of COVID-19 patient with aPL and arterial ischemia was described at the end of April 2020 by Chinese Authors [65] . Moreover, a high rate of Lupus Anticoagulant positivity, but not of the other aPL tests (anti-cardiolipin and anti-Beta2 glycoprotein I) was reported [66] . A larger multicentric cohort systematically assessed classic aPL tests together with 'non-criteria' aPL tests demonstrated a low rate of aPL positivity, as defined by classification criteria, and supported the idea that aPL found in COVID-19

patients are different from aPL found in antiphospholipid syndrome [67] . Further studies are necessary to clarify this issue.

Heparin and its derivatives are an under-exploited antiviral drug class, despite possessing broad-spectrum activity against a multitude of distinct viruses, including SARS-associated Coronaviridae [68] .

Even though the ACE2 protein is required for SARS-CoV2 entry, it was hypothesized that it was not the primary binding site on the target cell surface [68] . In fact, binding of the virus with heparan sulphates is probably required in the earlier phase of the interaction with human cells, as demonstrated for other human Coronaviridae [68, 69] .

Heparin is a well-known anticoagulant drug and is extensively used in clinical practice for its anticoagulant function in binding and activating antitrombin (AT). Over the treatment of venous thromboembolism, it is widely prescribed in a prophylactic setting in general medical inpatients, as well as in COVID-19 patients, according to the current guidelines for the clinical management [9] Since both heparin and heparan sulphate are complex, linear and acidic polysaccharides, belonging to the glycosaminoglycan (GAG) family, a possible competition in coronavirus binding was supposed.

A recent study demonstrated that SARS-CoV-2 surface protein S1 receptor binding domain binds to heparin and afterward induce a significant structural change [68] . Further proofs of the inhibitory potential of heparin against coronavirus infection come from in vitro studies, in which the incubation of susceptible epithelial cells (Vero cells) with heparin 30 min before SARS-CoV injection curtailed infection by 50% [70] .

An increasing number of cytokines and interleukins, such as IL-6, are now known to bind to GAGs of the heparin and heparan sulphate family. This binding allows the retaining of cytokines close to their site of release by the tissues in a mechanism of local regulation, thus enhancing their paracrine functions [71] .

Therefore, this could be considered as another potential mechanism by which heparin could interfere with the systemic cytokine storm detected in COVID-19. 

Dypiridamole (DIP) is an anti-platelet agent and acts as a phosphodiesterase (PDE) inhibitor that increases intracellular cAMP/cGMP levels [72] . In the past, it was demonstrated that DIP was efficacious in inhibiting the positive-stranded RNA viruses' replication [73] , in suppressing inflammation [74] and in preventing acute injury and progressive fibrosis of the lung, heart, liver, and kidney [75] . A recent study demonstrated an additional therapeutic benefit for COVID-19 patients [76] . urgently needed to evaluate the most appropriate prophylactic strategy, to prevent thromboembolic events that dramatically increase the morbidity and the mortality of these patients, but also to assess its ancillary anti-inflammatory and anti-viral effects.

Regarding research proposals, SARS-CoV2 potential of inducing coagulation dysfunction could provide insights into the pathogenetic mechanisms of the virus, potentially addressed by specifically targeted treatment strategies.

-Coagulation dysfunctions is not infrequent in COVID-19 patients, especially those with the most severe forms yielding to lower survival rates.

-Increased levels of D-dimer is the most frequently reported coagulation abnormality; a minority of patients also have APTT and/or PT prolongation and variable degrees of thrombocytopenia.

-The increased rate of thrombotic and thromboembolic events is now well recognized in COVID-19

series.

-In the cytokine storm, that characterize the most severe forms of COVID-19, IL-6 seems to play a pivotal role also in inducing a hyper-coagulable status both at systemic and local levels.

-SARS-CoV2 specific mechanisms seems to play an additional role, by inhibiting the fibrinolysis via the renin-angiotensin system (RAS).

-Heparin prophylaxis should be virtually prescribed to all COVID-19 hospitalized patients, in the light of hyper-coagulation provided by the inflammation and the virus, but also of the frequent concomitant cardiovascular risk factors. -Results from clinical trials on anticoagulant and anti-platelet agents in COVID-19 are needed to assess the optimal protocol to be adopted in clinical practice and to explore in vivo anti-viral and anti-inflammatory properties of these compounds.

The Authors have no conflicts of interest to declare

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