key: cord-0692022-60cj2sgp
authors: McFadyen, James D.; Stevens, Hannah; Peter, Karlheinz
title: The Emerging Threat of (Micro)Thrombosis in COVID-19 and Its Therapeutic Implications
date: 2020-06-26
journal: Circ Res
DOI: 10.1161/circresaha.120.317447
sha: d44bddb6f05dad2d19e09ba647be9338559b0bda
doc_id: 692022
cord_uid: 60cj2sgp

The recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing global pandemic has presented a health emergency of unprecedented magnitude. Recent clinical data has highlighted that coronavirus disease 2019 (COVID-19) is associated with a significant risk of thrombotic complications ranging from microvascular thrombosis, venous thromboembolic disease, and stroke. Importantly, thrombotic complications are markers of severe COVID-19 and are associated with multiorgan failure and increased mortality. The evidence to date supports the concept that the thrombotic manifestations of severe COVID-19 are due to the ability of SARS-CoV-2 to invade endothelial cells via ACE-2 (angiotensin-converting enzyme 2), which is expressed on the endothelial cell surface. However, in patients with COVID-19 the subsequent endothelial inflammation, complement activation, thrombin generation, platelet, and leukocyte recruitment, and the initiation of innate and adaptive immune responses culminate in immunothrombosis, ultimately causing (micro)thrombotic complications, such as deep vein thrombosis, pulmonary embolism, and stroke. Accordingly, the activation of coagulation (eg, as measured with plasma D-dimer) and thrombocytopenia have emerged as prognostic markers in COVID-19. Given thrombotic complications are central determinants of the high mortality rate in COVID-19, strategies to prevent thrombosis are of critical importance. Several antithrombotic drugs have been proposed as potential therapies to prevent COVID-19-associated thrombosis, including heparin, FXII inhibitors, fibrinolytic drugs, nafamostat, and dipyridamole, many of which also possess pleiotropic anti-inflammatory or antiviral effects. The growing awareness and mechanistic understanding of the prothrombotic state of COVID-19 patients are driving efforts to more stringent diagnostic screening for thrombotic complications and to the early institution of antithrombotic drugs, for both the prevention and therapy of thrombotic complications. The shifting paradigm of diagnostic and treatment strategies holds significant promise to reduce the burden of thrombotic complications and ultimately improve the prognosis for patients with COVID-19.

T he rapidly evolving coronavirus disease 2019 (COVID- 19) global pandemic is one of the greatest public health challenges since the Spanish flu pandemic over 100 years ago. Since the discovery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, millions of cases have been diagnosed worldwide resulting in hundreds of thousands of deaths. With no current effective therapy or vaccine for COVID-19, the enormous number of cases and deaths due to COVID-19 is likely to continue to increase. In this regard, although the adverse effects of COVID-19 were initially considered to primarily affect the respiratory tract by causing pneumonia and acute respiratory distress syndrome (ARDS), it has now become apparent that COVID-19 is associated with a prothrombotic state, which can manifest as microvascular thrombosis, venous, or arterial thrombosis, the presence of which usually portends an adverse prognosis. Thus, there is an urgent need to understand how SARS-CoV-2 induces a prothrombotic state to develop effective therapeutic approaches for COVID-19-associated thrombosis. This review will detail the significant emerging clinical findings of the prothrombotic status of patients suffering from COVID-19. We then provide an overview of the inherent links between the immune response and thrombosis, and how these mechanisms may help explain the prothrombotic effects of SARS-CoV-2. Finally, we will use this data to explore available therapeutic approaches for the prevention and treatment of COVID-19-associated thrombosis. Given the relative lack of data to date demonstrating the precise mechanisms of the link between SARS-CoV-2 infection, thrombosis, and clinical outcomes, we also highlight experimental data from other viral infections and thromboinflammatory disorders that are likely relevant to SARS-CoV-2 infection. Further research is urgently required to clearly define how SARS-CoV-2 infection results in thrombotic complications and how this impacts the disease course and severity. Therefore, this review will hopefully spark further interest and research, and importantly raise awareness of the importance of anticoagulation in patients with COVID-19 for both the prevention and treatment of thrombosis.

Since the beginning of the 21st century, 3 beta coronaviruses have been found to cause severe respiratory illness in humans, including SARS-CoV, MERS-CoV (Middle Eastern respiratory syndrome coronavirus), and now SARS-CoV-2. Coronaviruses reside in many animal hosts and can adapt to different species, including humans. The proximal origin of SARS-CoV and SARS-CoV-2 is thought to be bats, while the reservoir host for MERS-CoV is the dromedary camel. [1] [2] [3] Previous data suggest that in order for coronaviruses to exhibit efficient zoonotic transmission, the virus must undergo natural selection in an animal host (ie, intermediary species) such that it acquires features affording tropism to human tissue. To date, the intermediate host for SARS-CoV-2 is not known, however, a closely related coronavirus to SARS-CoV-2 has been identified in Malayan pangolins. 4 Moreover, it remains to be established whether SARS-CoV-2 has acquired genomic changes within an intermediate animal host before the occurrence of human transmission, or whether a SARS-CoV-2 progenitor may have undergone genomic changes during undetected human-to-human transmission, ultimately resulting in the current pandemic. 3 In humans, these coronaviruses gain entry into host cells by way of their transmembrane spike (S) GP (glycoprotein), which comprises an S1 and S2 subunit (Figure 1 ). The S1 subunit is responsible for binding to the host cell receptor, and the S2 subunit assists with viral and host cell fusion. 1 While the MERS-CoV S GP binds to DPP-4 (dipeptidyl peptidase 4) expressed on epithelial tissue, both SARS-CoV-2 and SARS-CoV bind to human cells via the ACE-2 (angiotensin-converting enzyme 2) receptor. 5 In addition to having different amino acid residues within the RBD (receptor-binding domain) of the S protein compared to SARS-CoV, another novel structural feature of SARS-CoV-2, is the presence of a polybasic furin cleavage site, at the junction of S1 and S2. Cleavage of the S protein by cellular proteases, including furin and TMPRSS-2 (transmembrane protease serine 2), is speculated to play an important role in the infectivity and host range of SARS-CoV-2, although its functional significance regarding how this feature may mediate SAR-CoV-2 transmissibility and pathogenicity is yet be fully elucidated. 1 In the context of COVID-19-associated thrombosis, understanding how these structural differences of SARS-CoV-2 may relate to the prothrombotic phenotype of COVID-19 is likely to be of fundamental importance given the significantly higher rates of thrombosis observed in SARS-CoV-2, compared with SARS-CoV and MERS-CoV infections. 

An emerging threat of the COVID-19 pandemic is the propensity of SARS-CoV-2 to cause microvascular, venous, and arterial thrombosis, and thereby exacerbating organ injury. Patients with severe COVID-19 appear to have a hyperinflammatory response, which is linked to the development of ARDS and multiorgan failure. 6 However, it is now well appreciated that the innate immune response and thrombotic response are closely linked ( Figure 2 ) and accordingly in patients with severe COVID-19 there is a correlation with elevated acute phase reactants, such as fibrinogen and CRP (C-reactive protein), which may contribute to COVID-associated hypercoagulability. [7] [8] [9] Moreover, these biomarkers and acute phase reactants are associated with adverse clinical outcomes and increased severity of infection. 7, 9, 10 In addition, abnormal coagulation parameters, including prolonged prothrombin time and activated partial thromboplastin time, are found to be associated with SARS-CoV-2 gains entry to host lung epithelial cells by the binding of the transmembrane spike (S) glycoprotein to ACE-2 (angiotensin-converting enzyme 2). The S1 subunit of the S protein binds to ACE-2 and mediates viral attachment. Proteolytic cleavage of the S protein at the S1/2 junction by the proteases, furin, and TMPRSS-2 (transmembrane protease serine 2), facilitates viral entry. SARS-CoV-2 can also directly invade endothelial expressed ACE-2. Infected cells undergo pyroptosis leading to the release of danger-associated molecular patterns (DAMPs) and triggering the release of proinflammatory cytokines and chemokines. The activated endothelium upregulates the expression of VWF (von Willebrand factor) and adhesion molecules including ICAM (intercellular adhesion molecule)-1, α v β 3 , P-selectin and E-selectin leading to recruitment of platelets and leukocytes and complement activation. Neutrophils release neutrophil extracellular traps (NETS), causing direct activation of the contact pathway. Complement activation potentiates these mechanisms by increasing endothelial and monocyte tissue factor (TF), further platelet activation and amplifies endothelial inflammation, which increases production of proinflammatory cytokines from the endothelium including IL (interleukin)-1, IL-8, RANTES (regulated on activation, normal T-cell expressed and secreted), IL-6, and MCP (monocyte chemoattractant protein)-1. The hypoxic environment can induce HIFs (hypoxia-inducible factors) which upregulates endothelial TF expression. These mechanisms ultimately lead to the unchecked generation of thrombin, resulting in thrombus formation. The fibrin degradation product, D-dimer, which is a marker of coagulation activation, appears to be a strong prognostic marker associated with high mortality in patients with COVID-19. Circulation Research. 2020;127:571-587. DOI: 10.1161/CIRCRESAHA.120.317447 a higher mortality from COVID-19, demonstrating the significance of coagulation abnormalities in this population. 3 Plasma D-dimer measurement is emerging as a direct prognostic marker in COVID-19. In this regard, D-dimer, a fibrin degradation product released when plasmin cleaves cross-linked fibrin, appears to be higher in patients with severe COVID-19 when compared with nonsevere disease. [11] [12] [13] [14] [15] Subsequently, further studies have highlighted that patients who do not survive COVID-19 have an elevated D-dimer level, and D-dimer continues to increase during admission before death. 10, 12, 16, 17 

Epidemiological data have demonstrated that race and ethnicity influence the risk of developing venous thromboembolism (VTE), with Blacks found to have the highest rate of incident VTE when compared with other ethnic groups, and Asians and Pacific Islanders having a lower rate of VTE than Whites. 18, 19 The underlying mechanisms for these racial differences are likely complex and to date remain to be fully understood. Previous data have suggested that higher Factor VIII levels, VWF (von Willebrand Factor) levels and lower Protein C levels observed in African populations may account for some of the racial differences in thrombotic risk. 20 In addition, recent data have highlighted important racial differences in platelet reactivity to thrombin, with platelets from Black patients commonly exhibiting polymorphisms in the PAR (protease-activated receptor)-4 that are associated with a markedly enhanced aggregation response to thrombin. 21 Interestingly, however, studies evaluating Chinese and White populations have demonstrated that abnormal coagulation parameters are associated with severe In vascular homeostasis, the endothelium possesses anti-inflammatory and antithrombotic properties due to the expression of CD39, nitric oxide (NO), and prostacyclin in addition to the natural anticoagulants, TFPI (tissue factor pathway inhibitor), activated protein C, and thrombomodulin. In the setting of infection or inflammation, endothelial cells upregulate the expression of VWF (von Willebrand factor) and adhesion molecules such as ICAM (intercellular adhesion molecule)-1, α v β 3 , P-selectin and E-selectin, promoting the adhesion of leukocytes and platelets. Activated platelets release chemokines CXCL1, PF (platelet factor)-4, CXCL5, CXCL7, CCL3, RANTES (regulated on activation, normal T-cell expressed and secreted), and CCL7 to enhance leukocyte recruitment. Leukocytes interact with platelets via several receptor/ligand pairs. These include platelet P-selectin binding to its cognate receptor PSGL (P-selectin glycoprotein)-1 on leukocytes, GP-Ib on platelets interacting with Mac-1 on monocytes and neutrophils, and GPIIb/IIIa binding to SLC44A2/CTL-2 on neutrophils. Activated platelets release polyphosphate (polyP), which activates the contact pathway, and HMGB (high mobility group box)-1, which enhances monocyte recruitment and monocyte tissue factor (TF) expression, thereby amplifying thrombin generation by way of the TF pathway of coagulation. Neutrophils release neutrophil extracellular traps (NETs) which promote thrombosis via activation of the contact pathway and the binding and activation of platelets. Finally, complement activation leads to the recruitment of leukocytes and upregulates TF expression, amplifies platelet activation and upregulates endothelial expression of proinflammatory cytokines, including IL (interleukin)-1, IL-6, IL-8, and MCP (monocyte chemoattractant protein)-1. These mechanisms result in excess thrombin generation, which potentiates the activation of platelets, leukocytes, and endothelium via PARs (protease-activated receptors) and culminates in a fibrin clot. infection and poor prognosis from COVID-19 regardless of ethnicity. 10, 12, 16, 22 Additionally, as discussed below, rates of thrombosis in COVID-19 appear increased regardless of the ethnic group studied, but the recognized and striking differences between racial groups are important to consider in the context of the SARS-CoV-2 pandemic.

Current research has highlighted that rates of VTE in patients with COVID-19 appear markedly increased. Indeed, the cumulative incidence of VTE is reported to be between 25% and 49% of patients with severe COVID-19 with pulmonary embolism being the most common thrombotic complication. [23] [24] [25] Importantly, even with the use of VTE thromboprophylaxis, the risk of VTE appears to remain elevated. 24, 26 Interestingly, when compared with non-COVID ARDS, patients with COVID-19 ARDS have a substantially increased rate of pulmonary embolism diagnosis (2.1% versus 11.7%, respectively, P=0.008) and patients diagnosed with a thrombotic complication have more than a 5-fold increase in all-cause mortality. 25, 27 Recently, 2 autopsy series have revealed that in patients with COVID-19 who were not clinically suspected to have VTE ante mortem, deep vein thrombosis, or pulmonary embolism was a common finding at autopsy (reported in 7/12 [58%] and 11/11 [100%], respectively). 28, 29 Furthermore, a small study found massive pulmonary embolism to be the direct cause of death in over 30% of patients, highlighting the important interplay between COVID-19 and venous thrombosis. 29 

In addition to VTE, there are emerging reports that the rate of arterial thromboembolism is increased in patients with COVID-19. Indeed, several groups have now reported rates of acute ischemic stroke of between 3% and 5% in patients admitted to hospital with COVID-19 and, in addition, preliminary reports suggest that these ischemic strokes are affecting younger people without traditional cardiovascular risk factors. 24, 30, 31 Furthermore, acute myocardial injury commonly complicates the clinical course of severe COVID-19 and is reported in up to 20% of hospital presentations with an adverse impact on mortality. 32 The underlying cause of myocardial injury is not yet evident, but it is speculated that in addition to potential damage from hypoxia or severe sepsis, SARS-CoV-2 may directly damage myocardial tissue by way of ACE-2 signaling, which is discussed further below. 33 As such, controlling for underlying cardiovascular risk factors in patients with COVID-19 remains a priority due to the association with increased severity of disease, in addition to a 5-fold increase in mortality in patients with underlying cardiovascular disease. 34, 35 

In addition to macrovascular complications, there is a strong association of COVID-19 with microvascular thrombosis. Pulmonary microvascular thrombosis has been previously described in autopsies as a complication of severe ARDS and has subsequently been reported to complicate ARDS during outbreaks of other coronaviruses, including SARS-CoV and MERS-CoV. [36] [37] [38] [39] [40] [41] However, this feature appears more pronounced in severe SARS-CoV-2 infection, with histology from patients with COVID-19-associated respiratory failure demonstrating a 9-fold increase in the prevalence of alveolar-capillary microthrombi when compared with patients with influenza. 42 In this regard, autopsy findings have shown that, in addition to the expected features of diffuse alveolar damage found in ARDS, platelet-fibrin thrombi are a common microscopic finding in the small pulmonary vasculature, occurring in 80% to 100% of lungs examined at autopsy. [43] [44] [45] In combination with other reported thrombotic events, the emerging microvascular thrombotic complications indicate a strong interaction between the SARS-CoV-2 and coagulation. Below, we review the underlying known mechanisms for thrombosis and the growing body of work that implicates SARS-CoV-2 as a key player driving many molecular processes underpinning pathological thrombosis.

The primary site of human infection with SARS-CoV-2 is the lung, where S1 attaches with high affinity to ACE-2 expressed on lung alveolar type II epithelial cells ( Figure 1 ). 1, 2, 5 The replication and release of virus from infected cells results in pyroptosis, a highly inflammatory form of cell death that is a common feature among many cytopathic viruses. PRR (Pattern recognition receptors), such as TLR (Toll-like receptors), subsequently detect the release of pathogen-associated molecular patterns, and intracellular danger-associated molecular patterns, such as ATP and DNA, which evokes an intense inflammatory reaction and triggers the release of proinflammatory chemokines and cytokines from neighboring cells ( Figure 1 ). 46 Strikingly, SARS-CoV-2 has been found to contain multiple single-stranded RNA fragments that can be recognized by TLR-7 and TLR-8, similar in numbers to MERS-CoV but more than SARS-CoV. 47 The recognition of SARS-CoV-2 by these receptors may contribute to innate immune hyperactivation and the cytokine storm often seen in severe COVID-19.

In the setting of SARS-CoV-2 infection, the vascular endothelium appears to be targeted directly by the virus Circulation Research. 2020;127:571-587. DOI: 10.1161/CIRCRESAHA.120.317447 as well as the milieu of proinflammatory cytokines elicited by the immune response. 48 ACE-2 is also expressed widely in extrapulmonary sites including blood vessels, heart, kidney, and intestine. 49, 50 Accordingly, viral RNA has been detected in urine and stool samples from patients with COVID-19. 51, 52 In this regard, it is currently hypothesized that viral entry via endothelial-expressed ACE-2 represents a mechanism by which the virus can enter and infect other tissues. Recent experimental data have highlighted the ability of COVID-19 to infect human endothelial cells. Indeed, human blood vessel organoids, which closely resemble human capillaries, contain viral RNA after exposure to COVID-19 in vitro. 53 Strikingly, the application of human recombinant soluble ACE-2 markedly inhibited viral infection in human capillary organoids, pointing to a direct role for endothelial ACE-2 in viral uptake in blood vessels. Intriguingly, the uptake of SARS-CoV-2 into cells is associated with the downregulation of ACE-2 expression. 54, 55 This appears to play an important role in promoting a proinflammatory and prothrombotic milieu since ACE-2 plays a pivotal role in regulating the RAS (renin-angiotensin system), by degrading angiotensin II to angiotensin 1-7. While angiotensin II induces vasoconstriction and promotes a proinflammatory/prothrombotic phenotype, angiotensin 1-7 opposes these effects via binding to the MAS receptor, which is widely expressed including on endothelial cells and platelets. 55 Interestingly, patients with SARS-CoV-2 have been demonstrated to have increased levels of IL (interleukin)-6, IL-1β, IFN (interferon)-γ, MCP-1 (monocyte chemoattractant protein 1), MIP (macrophage inflammatory protein), and IP10 (CXCL10). 11 These proinflammatory cytokines can disrupt endothelial function and integrity leading to the release of VWF, upregulation of adhesion molecules, such as ICAM (intercellular adhesion molecule)-1, integrin α v β 3 , P-and E-selectin, and the production of endothelial cytokines and chemokines. 56, 57 Thus, the endothelium adopts a proinflammatory and procoagulant phenotype that supports the recruitment of platelets and leukocytes. 58, 59 Furthermore, these changes are associated with the downregulation of the natural anticoagulants and corresponding upregulation of endothelial tissue factor (TF, CD142). 57 Moreover, a common finding in patients with COVID-19 is profound hypoxia. Although not formally tested in the context of SARS-CoV-2 infection, hypoxia is also likely to induce a prothrombotic endothelial phenotype since vascular hypoxia induces the activation of HIFs (hypoxia-inducible transcription factors) that have been demonstrated to upregulate endothelial TF expression while downregulating the natural anticoagulants, Protein S and TFPI (tissue factor pathway inhibitor), thus imparting a procoagulant endothelial phenotype. 60 A recent case series of autopsy sections from patients with COVID-19 aligns with these experimental findings described above and highlight the ability of COVID-19 to infect endothelial cells and cause endothelial injury. Sections of lung, kidney, and intestine from patients with COVID-19 demonstrate evidence of viral inclusion within endothelial cells, in addition to histological evidence of endothelial inflammation and cell death. 48 These data point to a direct role of COVID-19 in promoting systemic microvascular dysfunction which potentially promotes a prothrombotic state. 53 

Platelets are the second most abundant cell in the circulation with >1 trillion patrolling the vasculature at any one time. Therefore, in addition to their well-appreciated role in mediating hemostasis, they serve important immune functions. 61, 62 Platelets are equipped with a number of receptors and granular proteins affording them important roles in the immune response. Indeed, activated platelets release a number of chemokines from their alpha and dense granules that facilitate leukocyte recruitment to sites of vascular injury/inflammation. These include CXCL1, PF-4 (platelet factor 4/CXCL4), CXCL5, NAP-2 (CXCL7), and CCL3, RANTES (regulated on activation, normal T-cell expressed and secreted) (CCL5), and CCL7. 63 The release of these chemokines by platelets enhances leukocyte recruitment and adhesion to platelet thrombi and also serves to modulate leukocyte functional responses. Moreover, while lacking a nucleus, platelets express IL-1β mRNA that can be translated and secreted as mature IL-1β by activated platelets. 64 Further supporting the recruitment of leukocytes, platelets express a range of receptors that facilitate leukocyte adhesion. Activated platelets express P-selectin which allows leukocyte tethering via leukocyte expressed PSGL-1 (P-selectin glycoprotein ligand 1), while GP-Ib facilitates stable adhesion via binding to the leukocyte integrin Mac-1. 65, 66 More recently, platelets have been observed to have the unique ability to migrate, scavenge, and bundle bacteria up, thereby presenting them to neutrophils for phagocytosis and triggering the release of neutrophil extracellular traps (NETs), which can be induced by the P-selectin/PSGL-1 interaction as well as the recently described platelet GPIIb/IIIa (α IIb β 3 , CD41/CD61) interaction with SLC44A2 (CTL-2) on neutrophils. [67] [68] [69] Activated platelets further support coagulation by the release of inorganic polyphosphate (PolyP) from platelet dense granules which drives intravascular thrombin formation due to its ability to activate FXII. 70 Last, activated platelets release microvesicles, small particles ranging in size from 100 to 1000 nm, that are enriched in lipid and can carry a range of cargo including cytokines and chemokines, such as IL-1β, CXCL4, CXCL7, and CCL5, growth factors, microRNA, and mitochondria. 71 Moreover, CRP has recently been demonstrated to bind platelet microvesicles and activate complement via binding C1q. 72 Thus, the release of platelet microvesicles can regulate immune responses by mediating a range of target cells, particularly leukocytes and endothelial cells.

The most well-studied effect of SARS-CoV-2 on platelets pertains to its effect on platelet number. Thrombocytopenia has been reported as a common finding in severe COVID-19 with a recent meta-analysis demonstrating that thrombocytopenia was associated with a 5-fold increased risk of severe disease. 14, 16, 73, 74 The degree of thrombocytopenia in severe COVID-19 is usually mild and does not appear to be a common feature in nonsevere COVID-19. 73 Whether thrombocytopenia is a direct effect of SARS-CoV-2 or multifactorial in the setting of severe illness remains to be elucidated.

To date, there is a relative paucity of data regarding how SARS-CoV-2 may influence platelet function and potentially contribute to the prothrombotic phenotype observed in patients with COVID-19. However, there is now a large body of evidence demonstrating that viruses can directly interact with platelets thus potentially modulating their thrombotic and inflammatory function. 75, 76 Indeed, platelets are endowed with a large repertoire of adhesion receptors and immune receptors such as TLRs, FcγRIIa, and CXC/CCL receptors in addition to possessing the ability to engulf virions. Influenza, like SARS-CoV-2, is a single-stranded RNA virus which has been demonstrated to activate platelets via TLR-7 and the FcγRIIa receptor. The activation of platelet TLR-7 by viral RNA can evoke a range of functional responses which support thrombus formation, including platelet degranulation, the formation of platelet-leukocyte aggregates and platelet C3 release which in turn stimulates NETosis. [75] [76] [77] [78] In addition, previous work focusing on platelet activation by viruses has demonstrated that HSV-1 (herpes simplex virus 1) can induce thrombi via FcγRIIa on platelets and that the influenza strain H1N1 can activate platelets, both in a FcγRIIaand thrombin-dependent fashion, thus leading to platelet GPIIb/IIIa activation, arachidonic acid metabolism, and platelet microvesicle release. 79, 80 Corresponding mouse studies have emphasized an important role of platelets in H1N1 infection since mice infected with sublethal doses of H1N1 demonstrated large numbers of activated platelets in their lung tissue in addition to platelet-leukocyte aggregates and microvascular thrombosis. 81 Interestingly, inhibition of the major platelet adhesion receptor, GPIIb/IIIa, resulted in a protection from fatal influenza and a reduction in lung inflammation and microvascular thrombosis. 81 Moreover, recent studies have demonstrated that platelets from patients with influenza contain virus particles, while human platelets appear to have the ability to internalize the influenza virus. 77 Thus, a key outstanding area of research regarding the effects of SARS-CoV-2 and thrombosis pertains to how this virus interacts with platelets to mediate platelet number and function. In this regard, understanding if SARS-CoV-2 can be internalized and activate platelet TLR-7 and whether the ACE-2 receptor is expressed on platelets remain core unanswered questions. This is likely to be critical to understanding the link between COVID-19 and the prothrombotic phenotype given the key role of platelets in co-ordinating the thromboinflammatory response.

A prominent clinical feature of severe COVID-19 is the occurrence of respiratory decompensation typically 1 week after initial symptom onset. 82 This clinical trajectory appears to correlate with the time course of the adaptive immune response to SARS-CoV-2, with Tand B-cell responses to SARS-CoV-2 being detected ≈1 week after symptom onset. 46 Given it is postulated that this respiratory decompensation is driven by pulmonary microvascular thrombosis, understanding the intersection between thrombosis and the adaptive immune response to SARS-CoV-2 is of significant interest. These interactions are likely important since platelets have previously been demonstrated to be key modulators of adaptive immunity in the context of a range of inflammatory disorders. Platelets play an important role in regulating T-cell trafficking by facilitating the recruitment of T lymphocytes to sites of vascular injury or infection. 83 Moreover, platelets enhance T-cell functional responses via multiple mechanisms. For example, platelets express major histocompatibility complex class 1 molecules and can, therefore, directly activate T cells in an major histocompatibility complex class 1 dependent manner, 84 while platelet-derived PF-4 and serotonin can regulate Th17 expansion 85 and naïve T-cell activation and proliferation, 86 respectively. The importance of platelets in mediating T-cell recruitment and trafficking is underscored by data demonstrating that platelets play a fundamental role in the accumulation of cytotoxic T cells, and virusinduced organ damage, in a murine model of acute viral hepatitis. 87 Akin to the bidirectional relationship between platelets and other leukocyte subsets, the activation of T cells amplifies platelet aggregation with more recent data highlighting an important role for T cells recruited to sites of venous thrombosis in orchestrating the inflammatory response and subsequent thrombus resolution. 88, 89 In addition to mediating T-cell functions, platelets can increase B-cell immunoglobulin (Ig)G1, IgG2, and IgG3 production. 90 This is potentially significant in the setting of COVID-19 since the development of antiphospholipid antibodies, such as anti-β2 glycoprotein IgG antibodies, have been described in association with thrombosis in patients with COVID-19. 91 In addition, a cohort study from France describes a large percentage of patients with severe COVID-19 demonstrate a detectable lupus anticoagulant. 27 However, whether the presence of antiphospholipid antibodies in COVID-19 plays a role in COVID-19-associated thrombosis or represents merely an association remains to be established. Nevertheless, delineating how platelets modulate the adaptive immune response to SARS-CoV-2 and the role adaptive immunity plays in mediating thrombosis and organ injury will be crucial in uncovering novel therapeutic strategies.

The recruitment of leukocytes to a thrombus not only aids the immune response but also serves to facilitate platelet activation and coagulation. 92 Activated platelets facilitate neutrophils to release NETs, which are a matrix of DNA decorated with neutrophils granule proteins, such as MPO (myeloperoxidase), cathepsin G, and neutrophil elastase. 93, 94 NETs appear to be a primary defence mechanism to invading pathogens and as such have been demonstrated to trap and kill bacteria and fungi in addition to sequestering viruses. 94, 95 However, NETs have also been implicated in the pathogenesis of arterial and venous thrombosis in addition to a range of pathologies, including disseminated intravascular coagulation, sepsis, preeclampsia, vasculitis, and systemic lupus erythematosus. 96, 97 In the context of thrombosis, the release of the highly negatively charged NETs can trap erythrocytes and activate platelets. 98 Moreover, NETs serve as a scaffold and potent activator of coagulation via multiple mechanisms. Indeed, neutrophil elastase associated with NETs degrades the anticoagulant protein, TFPI, thereby enhancing TF activity and thrombin generation. 96 Histones activate platelets via TLR-2 and TLR-4 and enhance fibrin formation, while the contact activation system, via FXII activation, is initiated by negatively charged DNA. 96, 99 The activation of FXII not only initiates coagulation but also has important proinflammatory properties by way of its ability to liberate the generation of bradykinin, upregulate neutrophil function, and the generation of monocyte-derived proinflammatory cytokines. 100 Activated leukocytes, principally monocytes, and neutrophils, upregulate TF in response to proinflammatory cytokines, predominantly IL-6, and subsequently act to enhance coagulation. 101, 102 Additionally, monocytes provide a major source of intravascular TF by way of monocyte-derived, TFbearing microvesicles. 103, 104 The major danger-associated molecular pattern, HMGB (high mobility group box protein)-1 released by platelets during inflammation has also been shown to be modified by leukocytes, thus facilitating its ability to co-ordinate thrombosis. 105 Indeed, plateletderived HMGB-1 is oxidized by activated monocyte-derived reactive oxygen species, thereby imparting HMGB-1s prothrombotic potential acting in a paracrine fashion to enhance platelet activation, monocyte TF expression and NET formation, and ultimately thrombin generation. 105 Interestingly, it has been highlighted that type 1 interferons, a key family of widely expressed cytokines required for the co-ordinated innate immune response to bacterial and viral pathogens, enhance the release of HMBG1 thus enhancing the procoagulant activity of TF. 106 

The formation of NETs from activated neutrophils represents an innate immune response that aims to corral and kill invading pathogens. However, while having an important immune function, the Janus face of NETosis is the exacerbation of tissue injury, inflammation, and intravascular thrombosis. In the context of respiratory viruses, H1N1 infection has been linked to the excessive formation NETs in a mouse model of viral pneumonitis. 95 Moreover, the infiltration of neutrophils at sites of infection and the risk of ARDS development has been demonstrated in a range of other pandemic respiratory viruses, such as H1N1, SARS-CoV, and MERS-CoV. 107, 108 There is now data demonstrating that patients with COVID-19 exhibit elevated levels of NETs in serum as measured by cell-free DNA, MPO-DNA, and citrullinated histone H3. The levels of MPO-DNA and citrullinated histone H3 were higher in patients receiving mechanical ventilation compared with those not requiring any supplemental oxygen suggesting that levels of NETs correlate with disease severity. 109 Interestingly, the finding that the level of NETs correlated with elevations in D-dimer and sera from patients with COVID-19 could induce NETosis in purified neutrophils suggests that NETs, by way of their ability to generate thrombin, via activation of FXII, and activate platelets, may play an important role in mediating the prothrombotic phenotype observed in COVID-19.

One of the striking observations in severe COVID-19 is the correlation between inflammatory markers, such as CRP, ferritin and IL-6 and a raised D-dimer, the latter reporting on an activated coagulation pathway. Although a full discussion regarding the pathological hyperinflammatory response observed in severe COVID-19 is beyond the scope of this review, there is mounting evidence that monocytes/macrophages play a central role in this process. 104 This is perhaps best highlighted by the observation that patients with severe COVID-19 in ICU exhibit a significant expansion of CD14 + CD16 + monocytes producing IL-6 compared with nonhospitalized patients with COVID-19. As discussed above, this is particularly relevant in the context of thrombosis given the bidirectional relationship between monocytes and coagulation. First, activated platelets form heterotypic aggregates with monocytes in the setting of infection and inflammation. Platelet-monocyte aggregates may play a role in mechanical occlusion of the microvasculature in addition to altering platelet and monocyte functional responses. 110, 111 In this regard, platelets induce monocyte NF-κB (nuclear factor κ light-chain-enhancer of activated B cells) signaling and thereby modulate monocyte effector functions and induce the synthesis of the proinflammatory cytokines IL-8 and MCP-1. 112 Moreover, platelet P-selectin induces monocyte TF expression and upregulates the expression of monocyte Mac-1 (α M β 2 , CD11b/CD18). 113, 114 The platelet-derived chemokines, PF-4 and CXCL-12, enhance monocyte survival and facilitate differentiation into macrophages, 115, 116 while activated platelets also enhance the phagocytic activity of macrophages. 117 A consistent feature of COVID-19 is the finding that patients with existing cardiovascular risk factors are at an increased risk of severe COVID-19 and have higher fatality rates from the disease. 34, 35 It is noteworthy that oxidized phospholipids, which are strongly associated with atherosclerosis, have been detected in the lungs of patients with SARS-CoV. 118, 119 OxPLs (oxidised phospholipids) are generated by reactive oxygen species and play important roles in regulating inflammation given their demonstrated roles in promoting monocyte TF production, monocyte activation, and endothelial cell activation. 104 The oxidized phospholipid, oxPAPC, has been demonstrated to play a central role in the pathogenesis of a murine model of SARS-induced acute lung injury in a process linked to monocyte/macrophage activation via the TLR-4-TRIF-TRAF-6-NF-κB pathway. 119 

The complement cascade is another important arm of the innate immune system that has been implicated in COVID-19-associated thrombosis. Activation of the complement system, and vascular deposition of complement components, is linked to a range of thrombotic microangiopathies, including antiphospholipid syndrome and atypical hemolytic uremic syndrome. 120 Recent histological findings from biopsies obtained from patients with severe COVID-19 have demonstrated the extensive deposition of the terminal complement components, C5b-9 (membrane attack complex) and C4d, in addition to the MASP-2 (mannose-binding lectin-associated serine protease) within the microvasculature. 121 Strikingly, these vessels largely exhibited microvascular thrombi and, furthermore, the SARS-CoV-2 spike glycoprotein colocalized with C5b-9 and C4d in a subset of cases. 121 These findings appear consistent with a recent Chinese study where lung sections from patients with severe COVID-19 all revealed strong complement staining, while patients with severe COVID-19 have also been described to have elevated serum C5a levels. 122 Mechanistically, it has been suggested that the nucleocapsid (N) protein of SARS-CoV-2 binds to MASP-2 thereby triggering complement activation and amplifying tissue injury. The activation of the complement pathway leads to enhanced production of endothelial cytokines, such as IL-1, IL-8, RANTES, IL-6, and MCP-1, and also upregulates the expression of important endothelial adhesion molecules, such as P-selectin and VWF. 120, 123 Moreover, the anaphylatoxins C5a and C3a, act to facilitate the recruitment and activation of monocytes and neutrophils. Complement activation also directly induces a prothrombotic phenotype via C5a induction of neutrophil TF expression, C5b-7 monocyte TF expression, and C5b-8/C5b-9 mediated platelet activation. 124, 125 Furthermore, CRP has been described as a complement pathway activator and the correlation of its plasma level with COVID-19 severity highlights another potential link between immune and complement system. 72 These observations have attracted major interest in exploring the role of complement directed therapies in COVID-19 patients, in particular, in COVID-19 mediated thrombosis. 126 

The canonical role of thrombin is the enzymatic cleavage of fibrinogen to fibrin to ensure efficient hemostasis. However, thrombin also exerts pleiotropic proinflammatory effects on endothelial cells, platelets, and leukocytes ( Figure 3 ). In the context of viral infection, the coagulation cascade is activated as a host defence mechanism to try and limit pathogen spread. Indeed, a number of viruses have been demonstrated to directly activate coagulation, including HIV, Ebola, Coxsackie virus, and Dengue. 127 Previous data have highlighted that a major mechanism by which viral infection can induce a procoagulant phenotype pertains to the upregulation of TF expression by virus-infected endothelial cells and endothelial TLR-3 signaling in response to virus-derived pathogen-associated molecular patterns. 128, 129 However, excessive activation of coagulation and thrombin generation can result in deleterious consequences. Thrombin also signals via the PARs, which are widely distributed on a broad range of cells, including platelets, epithelial cells, immune cells, astrocytes, and neurons. 130 There are 4 members of the PAR receptors, PAR 1-4, all of which are G protein-coupled receptors. Thrombin efficiently cleaves and activates PAR-1, -3, and -4. 131, 132 Thus, in addition to being a potent platelet agonist, thrombin also activates immune effector and endothelial cells. 133, 134 Indeed, thrombin-induced activation of leukocytes results in the production of several cytokines and growth factors while also upregulating leukocyte adhesive function. Thrombin-induced endothelial activation, via PAR-1, induces the production of a range of proinflammatory cytokines and chemokines, including IL-6, IL-8, PAF (platelet-activating factor), and MCP-1, while also triggering the expression of the proadhesive molecules ICAM-1, E-and P-selectin. [133] [134] [135] Accordingly, PAR signaling has been demonstrated to modulate the immune response to a number of viruses. 127 In this regard, the inhibition of coagulation with an FVIIa/ TF nematode anticoagulant protein in a model of Ebola virus reduced D-dimer levels, intravascular thrombosis, IL-6, and MCP-1 concentrations and improved survival in treated animals. 136 Therefore, the disruption of the normal regulatory mechanisms governing thrombin production and activity occurs in the face of viral sepsis and the ensuing thromboinflammatory response. Thus, given the raised D-dimer levels observed in patients with severe COVID-19, it is likely that dysregulation of the thrombin production is a feature of the prothrombotic phenotype associated with SARS-CoV-2 infection.

The high rates of thrombotic complications observed in patients with severe COVID-19, combined with the current lack of an effective therapy for SARS-CoV-2, has led to significant interest regarding the use of antithrombotics for patients with COVID-19. In this regard, antithrombotic drugs which possess anti-inflammatory and/ or antiviral properties have already entered clinical trials and will be discussed below (Figure 4 ).

The strikingly increased prevalence of thrombotic complications has led to many hospitals instituting the routine administration of strict VTE prophylaxis with either low molecular weight heparin or unfractionated heparin and also resulting in several groups using intermediate or full-dose anticoagulation to prevent these thromboembolic phenomena. 137, 138 Preliminary reports have suggested that low molecular weight heparin treatment reduces mortality in COVID-19 patients with an elevated D-dimer or elevated sepsis-induced coagulopathy score. 139 The heparins are widely used anticoagulants which inhibit coagulation by the antithrombin mediated inhibition of FXa or thrombin. However, aside from their effects on anticoagulation, the heparins appear to have pleiotropic effects that may provide unique advantages in the context of viral infection, including anti-inflammatory effects by way of their ability to bind to dangerassociated molecular patterns, such as HMGB-1, and proinflammatory cytokines. 140, 141 Unfractionated heparin has a similar structure as heparan sulfate, the sulfated polysaccharide present on cell surfaces and extracellular matrix, with repeating disaccharide units. In contrast, low molecular weight heparin is a modified heparin with shorter polysaccharide chains. Intriguingly, heparan sulfate has previously implicated as a docking site for a number of viruses, thus facilitating cellular attachment of the virus and subsequent viral infection. 142, 143 Indeed, heparin, acting as a decoy receptor for naturally expressed heparan sulfate, has been shown to inhibit the binding of the SARS coronavirus to cells in vitro. 144 While ACE-2 is the major receptor that mediates viral entry into cells, heparan sulfate has previously been demonstrated to play an important role in facilitating coronavirus entry into cells by functioning as an adhesion receptor for the virus on the cell surface thereby allowing the increased density of viral particles on the cell surface which then facilitates ACE-2-mediated cell entry. 145 Recent studies have directly investigated the link between heparin and SARS-CoV-2 and suggest that heparin can directly bind with the spike protein of the SARS-CoV-2 virus and, therefore, may have antiviral properties. 146 Another interesting aspect of how the SARS-CoV-2 virus may be influenced by the use of heparin as anticoagulant is the previous observation that FXa can cleave the spike protein of SARS-CoV to render it more efficient in invading cells. 147 However, to date, this aspect remains to be investigated with SARS-CoV-2. These data have led some groups to propose specifically engineered heparins for the treatment of COVID-19, while clinical trials are underway to investigate the effects of low dose, and full-dose, anticoagulation with heparin on clinical outcomes for patients with COVID-19 (URL: https://www. clinicaltrials.gov. Unique identifiers: NCT04344756 and NCT04345848). 142 It is important to note that given the potential for heparin therapeutics to cause heparin-induced thrombocytopenia, but reassuringly, despite both thrombocytopenia and thrombosis being common complications of COVID-19, the development of heparin-induced thrombocytopenia appears limited to individual case reports. 148 

The inhibition of FXII as means to inhibit the thromboinflammatory response incited by severe COVID-19 appears an attractive and rational therapeutic target. Activation of the contact pathway is initiated by FXII activation by negatively charged molecules such as NETs, which are increased in severe COVID-19 patients, and plateletderived PolyP. 100 This leads to the generation of thrombin and the activation of bradykinin, of which the latter in particular results in downstream complement activation and the production of inflammatory cytokines. 149 Importantly, Heparins, including unfractionated heparin (UFH) and low molecular weight heparin (LMWH), bind antithrombin (AT), and potentiate the inhibitory effect of AT on coagulation factors Xa and thrombin. Furthermore, UFH may have antiviral effects by having the ability to bind the receptor-binding domain of the S protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in addition to potentially acting as a decoy for naturally expressed heparan sulfate thus reducing the ability of the virus to bind to and invade cells. The putative anti-inflammatory effects of UFH is related to its ability to bind danger-associated molecular pattern (DAMPs). Inhibitors of FXII block the contact factor pathway of coagulation, initiated by NETs, and also appear to have pleiotropic anti-inflammatory effects. Antiplatelet agents, such as dipyridamole, nafamostat, and aspirin inhibit platelet activation, which can inhibit NETosis and the release of platelet-derived DAMPs such as HMGB (high mobility group box)-1. Nafamostat may inhibit the TMPRSS-2 (transmembrane protease serine 2) and therefore impede viral entry. Fibrinolytics, such as tPA (tissue-type plasminogen activator), degrade cross-linked fibrin. therapeutic inhibition of FXIIa demonstrates protection from occlusive thrombus formation in animal models of thrombosis without impeding hemostasis. 150 Treatment with an inhibitory FXIIa antibody in animal models of systemic inflammatory response syndrome, using a lethal E coli challenge, have shown that FXIIa inhibition improves survival and decreases a number of inflammatory markers including complement activation, neutrophil degranulation, and IL-6. 151-153 Importantly, the potential safety of FXII inhibitors is highlighted by the fact that FXIIdeficient individuals display no bleeding phenotype, and there is no known effect on immune function. 154 A FXIIa inhibitory antibody has recently entered clinical trials for the treatment of hereditary angioedema and therefore represents a promising novel combination of antithrombotic and anti-inflammatory drugs (URL: https://www. clinicaltrials.gov. Unique identifier: NCT03712228).

Other therapies targeting coagulation are being examined regarding their potential therapeutic utility in COVID-19, including fibrinolytic agents such as tPA (tissue-type plasminogen activator). tPA converts the inactive enzyme plasminogen to plasmin leading to the subsequent breakdown of cross-linked fibrin. The interest in tPA has emerged from the concept that ARDS is characterized by significant local inflammatory reaction in addition to a hypofibrinolytic state. As discussed previously, severe COVID-19 can result in ARDS, which promotes mass upregulation of inflammatory cytokines and leukocytes, and can result in extensive thrombin generation, fibrin formation, and microvascular thrombosis. 155 The use of systemic tPA in preclinical and human models of ARDS demonstrates improved oxygenation, and a case report of tPA use in severe COVID-19 showed an initial improvement in oxygenation during tPA infusion, but this effect was lost after tPA therapy ceased. [156] [157] [158] However, the benefits of tPA may be offset by the considerable risk of major bleeding seen in nonstroke clinical trials, 159 which has led to the consideration of nebulized therapy to increase local concentrations and reduce systemic coagulation effects. Recent clinical trial data suggest that, in severe ARDS, nebulized fibrinolytic therapy is associated with improved oxygenation and ventilatory parameters, which may be another plausible treatment option in COVID-19. 155 

A recent meta-analysis has demonstrated that antiplatelet therapy is associated with improved mortality from sepsis. 160 Interestingly, the majority of data linking antiplatelet therapy with these improved clinical outcomes relates to aspirin. 161 In this regard, recent experimental data have highlighted that targeting platelets with aspirin prevents intravascular coagulation and neutrophilmediated microvascular thrombosis in a mouse model of bacterial sepsis. 162 However, to date, the effect of aspirin in COVID-19 remains to be established. Several other therapies with antiplatelet effects, including dipyridamole and nafamostat, are currently being evaluated for their potential role in reducing the severity of COVID-19. The antiplatelet agent dipyridamole has recently been reported to suppress SARS-CoV-2 replication in vitro, with some early data suggesting that use of adjunct dipyridamole may improve the clinical course in severe COVID-19. 163 Finally, the role of nafamostat, a serine protease inhibitor which has antiplatelet effects and is currently marketed in Asia for the treatment of disseminated intravascular coagulation and pancreatitis, is being evaluated. 164 Nafamostat is known to inhibit TMPRSS-2, and in preclinical studies, serine protease inhibitors are able to block SARS-CoV-2 infection of lung cells. Prior studies of nafamostat demonstrated that the drug has potential in blocking MERS-CoV infection in vitro, suggesting a potential role for this drug in reducing the burden of severe COVID-19. 5, 165 The RACONA Study (URL: https://www.clinicaltrials.gov. Unique identifier: NCT04352400) is a double-blind trial set to evaluate the efficacy of this agent in severe COVID-19. Nonetheless, it remains to be seen if any of these investigational therapies by virtue of their antiplatelet or antiviral effects will play a substantial role in the treatment of COVID-19.

The emergence of the SARS-CoV-2 virus and the ensuing global pandemic has presented unprecedented healthcare and economic challenges. A key clinical feature of severe COVID-19 seems to be a highly prothrombotic state, which is linked to excess rates of arterial, venous and microvascular thrombosis, and adverse clinical outcomes. Emerging evidence suggests that SARS-CoV-2 can infect endothelial cells with the associated immune response and ensuing activation of inflammatory pathways resulting in dysregulation of the endothelium, leukocyte activation, NET generation, complement deposition, and platelet consumption. These pathways can conspire to unleash a prothrombotic state (immunothrombosis), which may result in significant thrombotic complications. Accordingly, there remains much interest, and an urgent clinical need, to precisely delineate the mechanisms by which SARS-CoV-2 infection causes thrombotic complications in the hope that new insights into this process will yield novel therapeutic approaches. This is especially pressing in the absence of an effective therapy or vaccine for SARS-CoV-2. Thus, in the context of COVID-19 the intersection of innate and adaptive immunity, inflammation, and thrombosis has been thrust into the global spotlight, and the challenge is now to urgently find therapeutic concepts to disrupt this unholy trinity in the face of a global pandemic. The growing awareness and understanding of thrombotic complications in patients with SARS-CoV-2 infection will contribute to a more rigorous diagnostic approach resulting in the earlier detection of thrombotic events. Importantly, the shift towards the early institution of antithrombotic therapy for the prevention and treatment of COVID-19-associated thrombosis will hopefully translate into improved outcomes for patients with COVID-19. 

None.

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

A pneumonia outbreak associated with a new coronavirus of probable bat origin

The proximal origin of SARS-CoV-2

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

COVID-19 cytokine storm: the interplay between inflammation and coagulation

Prognostic value of interleukin-6, C-reactive protein, and procalcitonin in patients with COVID-19

COVID-19-associated hyperviscosity: a link between inflammation and thrombophilia?

Complex immune dysregulation in COVID-19 patients with severe respiratory failure

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

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China

Clinical characteristics of 140 patients infected with SARS-CoV-2 in Wuhan

China Medical Treatment Expert Group for Covid-19. Clinical characteristics of coronavirus disease 2019 in China

D-dimer is associated with severity of coronavirus disease 2019: a pooled analysis

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

D-dimer levels on admission to predict in-hospital mortality in patients with Covid-19

Incidence of venous thromboembolism in different ethnic groups: a regional direct comparison study

Effects of race and ethnicity on the incidence of venous thromboembolism

Racial differences in venous thromboembolism

Racial differences in human platelet PAR4 reactivity reflect expression of PCTP and miR-376c

COVID19 coagulopathy in Caucasian patients

Prevalence of venous thromboembolism in patients with severe novel coronavirus pneumonia

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

Confirmation of the high cumulative incidence of thrombotic complications in critically ill ICU patients with COVID-19: an updated analysis

Incidence of venous thromboembolism in hospitalized patients with covid-19

High risk of thrombosis in patients with severe SARS-CoV-2 infection: a multicenter prospective cohort study

Pulmonary arterial thrombosis in COVID-19 with fatal outcome: results from a prospective, single-center, clinicopathologic case series

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

Neurologic manifestations of hospitalized patients with coronavirus disease 2019 in Wuhan, China

Large-vessel stroke as a presenting feature of COVID-19 in the young

Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan

COVID-19 and the cardiovascular system

Prevalence of comorbidities and its effects in patients infected with SARS-CoV-2: a systematic review and meta-analysis

Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: Summary of a report of 72314 cases from the Chinese center for disease control and prevention

A clinicopathological study of three cases of severe acute respiratory syndrome (SARS)

The clinical pathology of severe acute respiratory syndrome (SARS): a report from China

Pulmonary pathology of severe acute respiratory syndrome in Toronto

Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore

Middle east respiratory syndrome coronavirus causes multiple organ damage and lethal disease in mice transgenic for human dipeptidyl peptidase 4

The pulmonary vascular lesions of the adult respiratory distress syndrome

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

Pulmonary and cardiac pathology in African American patients with COVID-19: an autopsy series from New Orleans

Pulmonary post-mortem findings in a series of COVID-19 cases from northern Italy: a two-centre descriptive study

Pathological evidence of pulmonary thrombotic phenomena in severe COVID-19

The trinity of COVID-19: immunity, inflammation and intervention

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV, and MERS-CoV genomes

Endothelial cell infection and endotheliitis in COVID-19

Angiotensinconverting enzyme 2 is an essential regulator of heart function

Angiotensin-converting enzyme II in the heart and the kidney

The presence of SARS-CoV-2 RNA in the feces of COVID-19 patients

Sars-cov-2 can be detected in urine, blood, anal swabs, and oropharyngeal swabs specimens

Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2

Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target

The pivotal link between ACE2 deficiency and SARS-CoV-2 infection

Elevated circulating E-selectin, intercellular adhesion molecule 1, and von Willebrand factor in patients with severe infection

The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome

Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin

Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm

The stimulation of thrombosis by hypoxia

Platelets and the immune continuum

Platelets are not just for clots

Thrombosis as an intravascular effector of innate immunity

Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis

Leukocyte accumulation promoting fibrin deposition is mediated in vivo by P-selectin on adherent platelets

Platelet glycoprotein ibalpha is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)

Migrating platelets are mechano-scavengers that collect and bundle bacteria

P-selectin promotes neutrophil extracellular trap formation in mice

Activated alphaiibbeta3 on platelets mediates flow-dependent netosis via slc44a2

Platelet polyphosphates are proinflammatory and procoagulant mediators in vivo

Platelet-derived microvesicles in cardiovascular diseases

Transitional changes in the CRP structure lead to the exposure of proinflammatory binding sites

Thrombocytopenia is associated with severe coronavirus disease 2019 (COVID-19) infections: a meta-analysis

Thrombocytopenia and its association with mortality in patients with COVID-19

Circulating platelets as mediators of immunity, inflammation, and thrombosis

Platelets and infection -an emerging role of platelets in viral infection

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

Platelet-TLR7 mediates host survival and platelet count during viral infection in the absence of platelet-dependent thrombosis

Influenza virus H1N1 activates platelets through FcγRIIA signaling and thrombin generation

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis

Severe covid-19

Platelets enhance lymphocyte adhesion and infiltration into arterial thrombus

Platelets present antigen in the context of MHC class I

Platelet factor 4 limits Th17 differentiation and cardiac allograft rejection

Serotonin provides an accessory signal to enhance T-cell activation by signaling through the 5-HT7 receptor

Platelets mediate cytotoxic T lymphocyte-induced liver damage

Platelet-lymphocyte conjugation differs between lymphocyte subpopulations

Innate effector-memory T-cell activation regulates post-thrombotic vein wall inflammation and thrombus resolution

Human platelets can activate peripheral blood B cells and increase production of immunoglobulins

Coagulopathy and antiphospholipid antibodies in patients with covid-19

The role of leukocytes in thrombosis

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

Neutrophil extracellular traps kill bacteria

Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases

Thrombosis: tangled up in NETs

Extracellular DNA traps promote thrombosis

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4

Coagulation factor XII in thrombosis and inflammation

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets

Blood-borne tissue factor: another view of thrombosis

Microparticles in hemostasis and thrombosis

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages

Disulfide HMGB1 derived from platelets coordinates venous thrombosis in mice

The role of type 1 interferons in coagulation induced by gram-negative bacteria

Immunopathogenesis of coronavirus infections: implications for SARS

A pathophysiologic approach to biomarkers in acute respiratory distress syndrome

Neutrophil extracellular traps in covid-19

Platelet-monocyte aggregates: bridging thrombosis and inflammation

Amicus or adversary: platelets in lung biology, acute injury, and inflammation

The phagocytes: neutrophils and monocytes

P-selectin induces the expression of tissue factor on monocytes

Effect of glycoprotein IIb/IIIa receptor blockade on platelet-leukocyte interaction and surface expression of the leukocyte integrin Mac-1 in acute myocardial infarction

Platelet-derived CXCL12 regulates monocyte function, survival, differentiation into macrophages and foam cells through differential involvement of CXCR4-CXCR7

The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages

Platelets mediate host defense against staphylococcus aureus through direct bactericidal activity and by enhancing macrophage activities

Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease

Identification of oxidative stress and Toll-like receptor 4 signaling as a key pathway of acute lung injury

Cross talk pathways between coagulation and inflammation

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases

Highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement over-activation

C5a-induced expression of P-selectin in endothelial cells

A novel C5a receptor-tissue factor cross-talk in neutrophils links innate immunity to coagulation pathways

Complement proteins C5b-9 stimulate procoagulant activity through platelet prothrombinase

Will complement inhibition be the new target in treating COVID-19-related systemic thrombosis?

Multiple roles of the coagulation protease cascade during virus infection

Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression

A key role for Toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells

Thrombin signalling and protease-activated receptors

Tissue factor-and factor X-dependent activation of protease-activated receptor 2 by factor VIIa

PARticipation in inflammation

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo

Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion

Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule

CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106)

Treatment of Ebola virus infection with a recombinant inhibitor of factor VIIa/tissue factor: a study in rhesus monkeys

Supported by the ESC Working Group on Pulmonary Circulation and Right Ventricular Function. COVID-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up: JACC State-of-the-art review

COVID-19 and its implications for thrombosis and anticoagulation

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure

More than an anticoagulant: Do heparins have direct anti-inflammatory effects?

Cell surface heparan sulfate and its roles in assisting viral infections

Herpesviruses and heparan sulfate: an intimate relationship in aid of viral entry

Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells

The 2019 coronavirus (SARS-cov-2) surface protein (spike) s1 receptor binding domain undergoes conformational change upon heparin binding

Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites

Heparin-induced thrombocytopenia with thrombosis in covid-19 adult respiratory distress syndrome

Roles of factor XII in innate immunity

A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk

The contact system contributes to hypotension but not disseminated intravascular coagulation in lethal bacteremia. In vivo use of a monoclonal anti-factor XII antibody to block contact activation in baboons

The contact activation system as a potential therapeutic target in patients with COVID-19

Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase

In vivo roles of factor XII

Fibrinolytic abnormalities in acute respiratory distress syndrome (ARDS) and versatility of thrombolytic drugs to treat covid-19

Tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ARDS): a case series

Meta-analysis of preclinical studies of fibrinolytic therapy for acute lung injury

Treatment of severe acute respiratory distress syndrome: a final report on a phase I study

PEITHO Investigators. Fibrinolysis for patients with intermediate-risk pulmonary embolism

Effect of antiplatelet therapy on acute respiratory distress syndrome and mortality in critically ill patients: a meta-analysis

Effects of antiplatelet therapy on the mortality rate of patients with sepsis: A meta-analysis

Acetylsalicylic acid inhibits intravascular coagulation during Staphylococcus aureus-induced sepsis in mice

Potential therapeutic effects of dipyridamole in the severely ill patients with covid-19

Potential of heparin and nafamostat combination therapy for COVID-19

Identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus S protein-mediated membrane fusion using the split-protein-based cell-cell fusion assay