Since the beginning of the pandemic, a very high incidence of thrombo-embolic events (VTE) was observed. The hypercoagulative state, described in patients with COVID-19 derives from a complex inflammatory response to the virus in which hemostasis and the immune system collaborate together to limit the spread of viral infection. Physiological immune thrombosis can evolve into an excessive, dysregulated formation of immunologically mediated thrombi and spread, especially in the microcirculation. Several viral infections may share abnormal coagulation processes such as bleeding, thrombosis, or both.

The increased incidence of VTE in COVID-19 patients was similar also in patients with other viral infections, i.e., severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS-CoV) ^[1][2][50,51]. H1N1 influenza infection is associated with an 18-fold increased risk of developing VTE when compared to critically ill patients with ARDS with no H1N1 influenza infection ^[3][6]. A previous study by Avnon et al. found that VTE occurred in 25% of patients with severe H1N1 influenza admitted to the intensive care unit (ICU) ^[4][52]. Particular evidence for thromboembolic events was also reported during cytomegalovirus (CMV) infection in which two arterial thrombotic events were described in nine Israelitic immunocompetent CMV-infected patients (spleen and liver) ^[5][53]. The pathophysiological mechanism is yet unknown but it seems to be related to higher levels of VWF in the plasma of CMV-infected people ^[6][54]. It is likely that the SARS-CoV-2 virus does not have intrinsic procoagulant effects, while coagulopathy appears as a consequence of the intense COVID-19 inflammatory response and endothelial activation/damage ^[7][55]. Two possible mechanisms implicated in the pathogenesis of coagulation dysfunction during SARS-CoV2 infection have been proposed: the cytokine storm which seems to play a pivotal role, and virus-specific mechanisms related to the virus interaction with the renin–angiotensin system and the fibrinolytic pathway ^[8][56].

Pro-inflammatory cytokines are involved in a so-called “cytokine release syndrome” responsible for the innate immune system activation and severe clinical manifestation of the disease ^[9][57]. Immune system dysfunction is a candidate risk factor for adverse outcomes in COVID-19, and the most important cause of morbidity and mortality in patients suffering from COVID-19 infection seems to be the cytokine storm causing an immune dysregulation in the peripheral tissues and in the lungs ^[10][58] (p. 2), refs. ^{[9][11][12][13][14]}[57,59,60,61,62].

Current evidence from clinical studies shows that IL-6 seems to play a prominent role in the cytokine-induced activation of coagulation. Additionally, IL-6 promotes the proliferation of megakaryocytes ^[15][64] and the release of TF, the latter detected in inflamed tissues and in particular in the lungs of patients affected by COVID-19 ^[16][65]. A postulated mechanism considers that SARS-CoV-2-infected megakaryocytes may interfere with platelet function and count, as already described in previous studies that reported thrombocytopenia during SARS-CoV infection. The virus induces the release of cytokines such as IL-6 conducting to megakaryocytic proliferation and differentiation, although the mechanism remains not completely clarified ^[17][18][66,67].

Furthermore, vascular permeability is mediated by IL-6 through the stimulation of vascular endothelial growth factor (VEGF) secretion and the release of other coagulation factors such as FIB and factor VIII ^[19][68]. There was a great effort during the pandemic to find inflammatory markers reflecting disease severity and eventually predicting disease prognosis. Among the most studied, increased levels of a pivotal serum cytokine, IL-1, which is a principal source of tissue damage interacting in both innate and acquired immunity, have been detected in patients suffering from severe COVID-19 infection. IL-1 stimulates the secretion of mediators stored in the granules of mast cells and macrophages, such as TNF-α, IL-6, and the release of arachidonic acid products such as prostaglandins and thromboxane A2 ^{[20][21][22][23]}[69,70,71,72]. Another important marker in the cytokine network of COVID-19 infection is IL-18. The catastrophic clinical course of COVID-19 shares similar features with macrophage activation syndrome (MAS) encountered also in other conditions with a potentially rapidly fatal course without treatment. IL-1, IL-6, IL-8, IL-10, IL-18, interferon (IFN)-γ, and TNF-α are the most important elements responsible for MAS development. IL-18 is produced by macrophages at very early stages of viral infections and induces the production of IL-6 and IFN-γ which are considered critical for optimal viral host defense. A study by Satis and coworkers observed a four-fold level of IL-18 in 58 people suffering from a severe form of COVID-19. These findings contrasted with the mildly affected patients and led to the conclusion of a correlation between IL-18 and the severity of the disease ^[24][73]. An additional role is determined by TNF-α, responsible for the activation of glucuronidases, which degrades the endothelial glycocalyx, and the upregulation of hyaluronic acid synthase 2, which leads to hyaluronic acid deposition and fluid retention ^[25][74]. Due to the systemic hypoxia induced by COVID-19-related ARDS, a reduction in endothelial nitric oxide synthase activity and nitric oxide levels has been indicated as a possible pathogenic process typical of endothelial dysfunction ^[26][75].

Experiments in vitro demonstrated that SARS-CoV2 can infect primary endothelial cells ^[27][76] and there is some evidence of the infection of endothelial cells in severe cases of COVID-19 ^[28][11]. Moreover, the replication within endothelial cells is able to induce cell death causing the activation of procoagulant reactions ^[29][77]. The membrane glycoprotein (Spike) of the SARS-CoV-2 virus interacts with Angiotensin-Converting Enzyme 2 (ACE-2), an integral membrane receptor expressed in the lung but also the heart, kidney, and intestine by reducing their activity. Normally, ACE-2 reduces the availability of angiotensin II through the counter-regulated activity of ACE ^[30][78].

The RAS may play a key role in SARS-CoV-2-induced COVID-19 ^[31][81]. The downregulation of ACE-2 by the virus causes an increase in angiotensin II, which, acting on the AT1 receptor, causes systemic injury ^[32][82] but also specific lung damage with pulmonary fibrosis, pulmonary inflammation, and ARDS in severe cases of COVID-19 ^[33][83]. ACE-2 is markedly expressed in pneumocytes type II, hence participating in alveolar surfactant production. The downregulation of ACE-2 receptors due to the binding of coronavirus might hinder the expression of pneumocytes type II cells, explaining the worsening of gaseous exchange ^[34][35][84,85]. Overall, the interaction of coronavirus with ACE-2 receptors is destructive due to increased inflammatory lesions, the downregulation of ACE-2 receptors, increased local angiotensin II effects and AT1 receptor over-activity, insufficient surfactant due to bruised pneumocytes type II causing a reduction in pulmonary compliance and amplified surface tension, and a reduction in the generation and repair of pneumocytes type I with impaired gaseous exchange along with alveolar–capillary diffusion capacity and fibrosis ^[36][86]. Moreover, a different impact of SARS-CoV-2 expression on ACE-2 may be due to gender-related dissimilarities, with the ACE-2 gene existing in the X-chromosome ^[37][87]. The wide variances in COVID-19 death rates might be explained by significant alterations in the equilibrium of the ACE:ACE-2 system associated with gender, racial, and age differences in genetic ACE and ACE-2 polymorphism and environmental aspects manipulating ACE-2 expression ^[38][39][40][88,89,90]. In addition, the severity of lung injury is linked with the expression of ACE. ALI was less complicated in complete knockout (Acee/e) mice and AT1 receptor knockout mice compared to partial ACE knockout (Ace./e) mice and wild-type mice, respectively. The injection of recombinant SARS spike protein along with AT1 blockers elevated the expression of angiotensin II leading to ARDS in mice ^[41][91]. Thus, understanding the role of the ACE-2 receptor in the pathogenesis of COVID-19 may open a potential approach for therapeutic intervention ^[42][92].

Among virus-related mechanisms, high levels of PAI-1, the principal inhibitor of fibrinolysis interfering with tissue plasminogen activator (tPA) and urokinase, have been related to an increased risk of thromboembolic events ^[43][80]. Interestingly, previous studies reported high blood levels of PAI-1 in patients with SARS-CoV infection suggesting a possible direct effect of infection on the production of anti-coagulant factors ^[44][93]. One study described an important increase in another mediator of platelet adhesion, platelet-derived vitronectin (VN), in SARS-CoV pneumonia; however, it was not possible to discriminate its origin from increased expression by the liver or from lung damage ^[45][94]. Another possible virus-specific effect could be related to the induction of autoimmunity, also described in SARS patients ^[46][37]. Recent studies showed that the appearance of antiphospholipid antibodies and lupus anticoagulant immunoglobulins may have a role in the pathogenesis of coagulopathy. Indeed, the presence of IgA anti-cardiolipin antibodies and IgA and IgG anti-2-glycoprotein I antibodies have been found in association with coagulopathy, thrombocytopenia, and the development of peripheral and cerebral ischemic events. Antiphospholipid antibodies (aPL), recognized as risk factors for arterial and venous thrombosis, have been associated with different viral infections, such as parvovirus B19, herpes viruses, hepatitis viruses, and human immunodeficiency viruses. The first case report of a COVID-19 patient with aPL and arterial ischemia was described by Chinese authors ^[47][95], although, subsequently, a larger, multicentric cohort demonstrated a low rate of aPL positivity, as defined by classification criteria, suggesting that aPL found in COVID-19 patients is different from aPL found in antiphospholipid syndrome ^[48][96]. It is likely that the mechanisms of altered coagulation due to SARS-CoV-2 infection, also responsible for hypoxia, may in turn favor the thrombo-inflammatory loop and consequently increased blood viscosity and the release of procoagulant antibodies ^[49][32]. These observations were confirmed by a study by Harzallah and coworkers investigating 56 patients with confirmed or suspected SARS-CoV-2 infection. Among these, 25 were found with lupus anticoagulant immunoglobulin, whereas 5 were found positive for IgM or IgG anti-cardiolipin or anti-2-glycoprotein I antibodies ^[50][33]. Further studies are needed to address this issue.

COVID-19-related coagulopathy firstly determines elevated D-dimer levels that combine in turn with mildly prolonged PT, APTT, and mild thrombocytopenia. At late stages, this process evolves into a classical DIC ^[51][97]. These findings were identified in the clinical setting in a meta-analysis where 7.613 patients suffering from COVID-19 infection were examined. In this cohort, thrombocytopenia was worse in the critically ill group than in those with non-severe disease ^[52][98]. Additionally, the platelet count was lower in the elderly, in males, and in patients with higher APACHE II scores at admission ^[53][99]. This study highlights an association between low platelet counts and an increased risk of severity of the disease and mortality. As per SARS-CoV-2-infection-related thrombocytopenia, it appears that the platelets can be more rapidly removed or sequestrated by the reticuloendothelial system after the activation of antigen–antibody complexes ^[54][55][100,101]. Additionally, the megakaryocyte’s function and the consequent platelet production can be reduced by the virus activity ^[56][102]. A possible mechanism of thrombocytopenia was described after COVID-19 vaccination. It was observed in rare cases that immune thrombotic thrombocytopenia (VITT) syndrome was induced by the vaccine, particularly the ChAdOx1 nCoV-19 vaccine. The main pathogenetic hypothesis supporting this evidence is the possible promotion of antibody synthesis against PF4 by some anti-COVID vaccines promoting the synthesis of antibodies against PF4 that provoke platelets’ massive activation, inducing immune thrombotic thrombocytopenia ^[57][103]. As anti-PF4 antibodies were detected in patients with VITT, the current guidelines recommend a PF4-heparin ELISA blood test before performing a vaccine when VITT is clinically suspected ^[58][104]. The risk of clotting in the general population is estimated to be around 1:250,000, although it is higher in young people (20–29 years old) at 1.1:100,000 ^[59][105].

Sepsis is a life-threatening condition as a response to a primary infection in which the body responds with extreme inflammatory reactions that create injuries in one’s own tissues and organs. On the other hand, severe COVID-19 infection is commonly complicated with coagulopathy, and, in the latter stages, may evolve towards a classical DIC. These manifestations were an object of major concern during the COVID-19 pandemic. The International Society of Thrombosis and Hemostasis (ISTH) has proposed a new category to identify an early stage of DIC associated with sepsis called sepsis-induced coagulopathy (SIC). Many patients suffering from severe COVID-19 meet the Third International Consensus Definitions for Sepsis (Sepsis-3) ^[60][106] manifesting respiratory dysfunction during a viral infection.

In the setting of the altered coagulation state, the measurements of the coagulative parameters may orient the clinicians toward the early identification of a coagulative derangement. Besides the D-dimer, as above mentioned, other parameters are of bedside interest. Increased levels of thrombin–antithrombin complexes, plasmin-alpha-2-antiplasmin, and thrombomodulin complexes have been reported in respiratory tract infections. Increased PAI-1 serum levels were identified, suggesting impaired fibrinolysis. A study ^[61][15] highlighted an alteration of the laboratory parameters deponent for DIC (according to the diagnostic criteria of the ISTH) in 15 subjects (71.4%) who died of COVID-19-related pneumopathy. In the final stage of the disease, elevated levels of D-dimer and FIB degradation products were found. Recent contributions have reported that COVID-19 severity could be associated with some coagulopathy biomarkers, including prothrombin time (PT), activated partial thromboplastin time (APTT), and D-dimer. Nevertheless, the association between coagulopathy and COVID-19 severity still remains undefined.

The severity of the condition is mostly associated with clinical evidence. In particular, one study ^[62][109] demonstrated that the majority of patients developed a mild infection, and about 15% of them experienced a severe manifestation with dyspnea and hypoxia. Another 5% developed respiratory failure in conjunction with ARDS, shock, and multi-organ dysfunction. Many studies have focused on the evaluation of D-dimer, PLT, PT, APTT, and FIB. It was reported that D-dimer and PT values have been shown to be higher in patients with more severe disease ^[63][110]; moreover, several studies have shown that elevated D-dimer levels are associated with in-hospital mortality. Recent research studies have hypothesized that genetic profiles may partly explain individual differences in developing thrombotic complications during COVID-19 infection. An interesting study evaluated the genotypic distribution of targeted DNA polymorphisms in COVID-19 complicated by pulmonary embolism during hospitalization, finding significant associations between higher D-dimer levels and ACE I/D and APOE T158C polymorphism in patients with and without pulmonary embolism, suggesting a potentially useful marker of poor clinical outcomes ^[64][111]. Previous data showed a higher prevalence of ACE D/D genotype in severe COVID-19 patients compared to those with mild disease; this genotype is significantly associated with cardiometabolic diseases and obesity, known risk factors for COVID-19 ^{[65][66][67][68]}[112,113,114,115]. Additionally, this genotype was associated with thrombo-embolic manifestations in patients affected by other diseases and traditional thrombophilia-related polymorphisms ^[69][116], increased venous thromboembolism risk ^[70][71][117,118], and endothelial damage with hypercoagulability in patients with arterial hypertension ^[72][119]. The APOE locus has been associated with increased vulnerability to severe COVID-19 mortality, especially for the APOE4 homozygous genotype ^[73][120] which is the strongest genetic risk factor for sporadic Alzheimer’s disease. This appears to be very important from a clinical point of view as recent data show that dementia can predict the severity of COVID-19 infection. In fact, patients with dementia are more exposed to the severe form of the infection and are more likely to require hospitalization and to have severe sequelae or fatal outcomes compared with patients who do not ^[74][75][5,121]. Finally, the racial variance of ACE I/D genotype polymorphism seems to be correlated with different outcomes during COVID-19 infection; in fact, populations with higher D allele frequency (e.g., Italian) experienced higher fatality ^[76][122]. In another meta-analysis, it was demonstrated that the platelet count decreased progressively with the degree of disease severity ^[77][123]. However, a previous meta-analysis ^[78][124] demonstrated that there were no differences in PLT and APTT levels between wild and severe cases. All this is probably due to the confounding factors and biases that inevitably occur, such as age, sex, and the presence of comorbidities such as hypertension, diabetes, cardiovascular disease, and chronic kidney disease of the examined populations. As reported in another study by Wu et al., mortality from severe COVID-19 was increased 34-fold compared to a normal infection ^[79][125] and very high levels of coagulation markers were correlated with an 11-fold increase in death. These observations underline the importance of the early stratification of disease severity.

Data on anticoagulant therapy appear to be associated with a better outcome in moderate-to-severe COVID-19 patients with altered coagulative parameters (elevated D-dimer, elevated FIB, and low levels of anti-thrombin) ^[80][81][82][13,14,132]. A retrospective study by Shi et al. showed that these treatments can mitigate cytokine storm exerting an anti-inflammatory effect (reduction in IL-6 and increase in lymphocytes) and improving coagulation dysfunction ^[83][133]. A number of substances are used for COVID-19 VTE such as heparins, direct oral anticoagulants (DOAK), aggregation inhibitors, factor XII inhibitors, thrombolytic agents, anti-complement, anti-NET drugs, and IL-1 receptor antagonists.

Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH), have several anti-coagulant and anti-inflammatory effects ^[84][134]. Among the various properties of heparin, a beneficial effect on endothelium has been observed. Dysfunctional endothelium leads to an inflammatory status through the production of vasoconstrictor factors and the recruitment of immune cells ^[85][135]. Histones released from damaged cells may be responsible for endothelial injury ^[86][136]. Heparin exerts its action through an effect on histone methylation and MAPK and NF-κB signaling pathways ^[87][137]. In this way, heparin can antagonize histones and therefore “protect” the endothelium ^[88][89][29,30]. It was proved to have a beneficial effect related to its anticoagulant function on COVID-19 ^[90][138] and anti-inflammatory properties ^[91][139]. The proposed mechanisms include binding to inflammatory cytokines, the inhibition of neutrophil chemotaxis and leukocyte migration, the neutralization of complement factor C5a, the sequestration of acute-phase proteins such as P-selectin and L-selectin, and the induction of cell apoptosis through the TNF-α and NF-κB pathways ^[92][93][140,141]. Another potential direct antiviral role of heparin is related to its polyanionic properties allowing it to bind to various proteins thus acting as an effective inhibitor of viral adhesion ^[94][142]. This condition mechanism was also described in other viral diseases ^[94][95][142,143] as well as in SARS-CoV. As Mycroft-West et al. ^[96][144] demonstrated, surface plasmon resonance and circular dichroism were used, and it was demonstrated that the receptor binding domain of the Spike S1 SARS-CoV-2 protein interacts with heparin. In a report by Tang ^[61][15], a favorable outcome was highlighted with the use of LMWHs in severe patients with COVID-19 who meet the criteria of SCI (sepsis-induced coagulopathy) or with markedly elevated D-dimer. A large, retrospective multicentric study among in-hospital patients (the CORIST study) showed that heparin treatment was associated with lower mortality, particularly in severely ill COVID-19 patients and in those with strong coagulation activation ^[97][145]. Moreover, research conducted in the neurorehabilitation department of a neuroscience referral hospital following neurological damage showed, despite a small number of patients, that hospitalized, vulnerable, patients with severe neurological damage can present a completely unexpected benign disease course of SARS-CoV-2 infection after heparin treatment. The anti-inflammatory and anticoagulant effects of enoxaparin administered much earlier before and during the infection, together with possible antiviral activity, could explain the favorable disease course observed in severe neurological patients with an increased risk of poor outcomes. Further research is needed to explore the possible mechanisms of action of enoxaparin in critical neurological patients with COVID-19 and confirm these observations ^[98][146].

The contact activation system, including factor XII (FXII), factor XI (FXI), high-molecular-weight kininogen, and prekallikrein, links inflammation and coagulation, triggering thrombin generation which promotes platelet activation but also upregulates the kallikrein–kinin system (KKS) which induces the renin–angiotensin system with the release of pro-inflammatory cytokines ^[99][155]. The inhibition of contact activation has been shown, especially in animal models, to prevent consumptive coagulopathy, pathologic systemic inflammatory response, and mortality ^[100][156]. Direct FXa inhibitors have been already shown to possess an inflammatory and antiviral effect in addition to their well-established anticoagulant activity, and they have been proposed to have a potential therapeutic role in coronavirus infections ^[101][157]. FXI activation by virtue of its position as an interface between contact activation and thrombin generation has been suggested as a unique and promising target to safely prevent or treat COVID-19-related inflammatory complications including cytokine response and coagulopathy, hence reducing associated mortality, and, evidence from recent research suggests that the inhibition of FXIa seems to attenuate thrombosis with little effect on hemostasis and may also have a potential role on infections ^[102][158]. Direct inhibitors of FXIa using small peptidomimetic molecules, monoclonal antibodies, aptamers, or natural inhibitors have been developed in recent years ^[103][159]. Preclinical data and rationale exist for preventing the activation of FXI and FXII preserving the hemostatic activity of FXI in COVID-19, and several inhibitors of FXII and FXI are currently under investigation ^[102][158] representing a promising therapeutic target against COVID-19 patients with severe disease.