The Emerging Threat of (Micro)Thrombosis in COVID-19 and Its Therapeutic Implications

Abstract

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.

Keywords: coronavirus; mortality; stroke; thrombosis; viruses.

Figure 1.

Proposed mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, and coronavirus disease 2019 (COVID-19)-associated thrombosis. 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_vβ₃, 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.

Figure 2.

Mechanisms regulating immunothrombosis. 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_vβ₃, 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.

Figure 3.

Proposed role of thrombin in coronavirus disease 2019 (COVID-19)-associated immunothrombosis. Infection of endothelial cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and liberation of viral danger-associated molecular pattern (DAMPs) results in endothelial activation with the upregulation of tissue factor (TF) and adhesion molecule expression in addition to endothelial cytokine production. This leads to the recruitment and activation of leukocytes and platelets. Activated leukocytes release neutrophil extracellular traps (NETs) and monocyte-derived, TF-bearing microvesicles (MV). Activated platelets release polyP from dense granules. This initiates intravascular thrombin generation via the TF and contact pathways of coagulation. Thrombin exerts its thrombotic effect by activating platelets through the platelet PAR (protease-activated receptor)-1/4 in addition to mediating the cleavage of fibrinogen to fibrin. Furthermore, thrombin possesses proinflammatory functions due to its ability to activate endothelial cells and leukocytes. Thrombin activates endothelial cells via the endothelial PAR-1 receptor, leading to upregulation of IL-6, IL-8, PAF (platelet-activating factor), and MCP (monocyte chemoattractant protein)-1 in addition to the adhesion molecules P-selectin, E-selectin and ICAM (intercellular adhesion molecule)-1, all of which serve to increase leukocyte recruitment and activation. Similarly, monocyte and T-cell functions are enhanced by thrombin activation of monocyte and T-cell expressed PAR-1 and PAR-3. The resultant endothelial cell, platelet, and leukocyte interactions establish a positive feedback loop, which further promulgates ongoing thrombin generation leading to immunothrombosis. This thrombotic phenotype likely results in the clinical manifestations seen in COVID-19, including pulmonary embolism (PE), microvascular thrombosis, ischemic stroke, and deep vein thrombosis (DVT).

Figure 4.

Treatments for targeting coronavirus disease 2019 (COVID-19)-associated thrombosis. 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.