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 (McFadyen et al, Circulation Research, 2020). Studies from the Netherlands and France suggest that clots arise in 20–30% of critically ill COVID-19 patient (Poissy et al, Circulation, 2020) Given thrombotic complications are central determinants of the high mortality rate in COVID-19, strategies to prevent thrombosis are of critical importance (McFadyen et al, Circulation Research, 2020).
It is for its anti-thromboxane, anti-platelet action that aspirin is used in patients with heart disease to prevent clotting in the arteries of the heart. However, aspirin works by blocking an enzyme in platelets called COX-1 but aspirin is not very effective in blocking the production of thromboxane A2 from endothelial cells when these cells are injured as happens in COVID-19. This is because endothelial cells produce thromboxane A2 by the action of COX-2, and aspirin does not block COX-2 effectively. However, aspirin is currently being used in COVID-19, as was in the case of the U.S. president, but there is no evidence so far that it is effective in the disease. Instead of targeting the enzymes that produce thromboxane A2 a more elegant approach maybe to block the receptor for thromboxane A2. This would allow for continued production of beneficial prostaglandins such as PGI2, also referred to as prostacyclin. In fact, blocking COX-2 using drugs such as Celebrex is potentially risky because in addition to blocking thromboxane A2 production it will also block prostacyclin production thereby increasing the risk of blood clotting. It is notable that adiposity and aging strongly associate with first, severity of COVID-19, and second, aspirin resistance due to increased generation of thromboxane A2. Ramatroban holds potential advantages over aspirin, especially in COVID-19 patients: first, ramatroban is 100 times more potent that aspirin as an antiplatelet and antithrombotic agent (Karyazono et al, Blood Coagulation and Fibrinolysis, 2004); second, unlike aspirin, ramatroban’s antiplatelet effect is reversible and this is a significant advantage if bleeding ensues (major bleeding occurs in > 5% of patients with severe COVID-19)15; and third, ramatroban should be effective in the elderly and the obese since it blocks COX-2 mediated thromboxane A2, while low-dose aspirin only inhibits COX-1.
TP receptor activation on platelets leads to P-selectin expression (Matsui et al, 2012), which is markedly increased in COVID-19. P-selectin mediates inflammation and notably formation of neutrophil extracellular traps (NETs). NETs, which are released from activated neutrophils, protect against infection, in particular by large pathogens, but they are also implicated in the pathology associated with a growing number of immune-mediated conditions. In others words, NETs are geared more toward fighting a worm rather than a virus. However, microvascular thrombi containing neutrophils, platelets, and NETs are a hallmark of severe acute respiratory syndrome corona virus 2 infection, linking multiorgan failure and systemic hypercoagulability in COVID-19 (Nicolai et al, Circulation, 2020). A marker of NET formation of the release is myeloperoxidase. In a rat model of endotoxic shock, ramatroban prevented hypotension, reduced plasma TNF levels by over 90%, and markedly reduced myeloperoxidase levels in lungs, ileum and heart, suggesting end-organ protection by mitigating thromboxane A2 mediated platelet-polymorphonuclear leukocyte activation, and improved survival by 45% (Altavilla et al, Pharmacol Res, 1994). In rats with splanchnic artery ischemia-reperfusion injury, while plasma levels of thromboxane B2 were increased about 7-fold, ramatroban prevented hypotension, improved survival, restored phagocytic function of peritoneal macrophages partially, inhibited plasma myocardial depressant factor activity about 50%, and inhibited tissue infiltration by neutrophils as measured by decline in ilium myeloperoxidase activity > 50% and lung myeloperoxidase activity > 80% (Canale et al, Pharmacology, 1994). Therefore, Ramatroban will effectively inhibit the formation of NETs in COVID-19 by blocking thromboxane A2 mediated P-selectin expression on platelets.
Third, TP receptor activation on endothelial cells leads to expression of adhesion molecules, which mediated immunosurveillance, inflammation, hemostasis, wound healing, morphogenesis, maintenance of tissue architecture, atherogenesis, and tumor metastasis. Ramatroban improves vascular responsiveness; while inhibiting endothelial surface expression of ICAM-1 and VCAM-1; inhibiting MCP-1 expression in response to TNF-α or platelet activating factor; and inhibiting macrophage infiltration (Ishizuka et al, Cardiovasc Drug Rev., 2004)
Dengue virus activates platelets via CLEC-2, leading to release of extracellular vesicles including exosomes and macrovesicles, which activate CLEC-5a and toll-like receptor 2 (TLR2) on neutrophils and macrophages, thereby inducing neutrophil extracellular trap (NET) formation and proinflammatory cytokine release (Sung et al, Nature Communications, 2017). Upon activation by dengue virus, CLEC-2 receptor undergoes tyrosine phosphorylation mediated by thromboxane A2. This leads to downstream phosphorylation of spleen tyrosine kinase (Syk) and phospholipase γ2 (PLCγ2) phosphorylation, again potentiated by thromboxane A2 (Badolia et al, JCB, 2017). This cooperation between CLEC-2 and thromboxane A2 signaling is critical for platelet activation since ramatroban (Bay u 3405), a potent thromboxane A2 receptor antagonist completely blocks CLEC-2 signaling and thereby inhibits platelet activation. In this regard, it is notable that the levels of plasma thromboxane B2 levels are markedly increased in COVID-19 patients relative to healthy controls (Archambault et al, MedRxiv, 2020) The potential role of CLEC-2 in mediating SARS-CoV-2 induced platelet activation merits investigation in order to help design optimal strategies to prevent and treat immune-thrombosis in COVID-19.
(Sung and Hsieh, Frontiers in Immunology, 2019)
(Badolia et al, JCB, 2017)
Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology (Fig. 1). Activation of sC5b-9 (membrane attack complex) and C4d (classical lectin pathway) are associated with respiratory failure in hospitalized patients with COVID-19 (Holter et al, PNAS, 2020). sC5b-9 plasma levels are also elevated in children with SARS-CoV-2 infection, even if they have minimal symptoms of COVID-19 (Diorio et al, Blood Advances, 2020). 13 of the 34 children with available peripheral blood smears met clinical criteria for thrombotic microangiopathy associated with complement activation. Median D-dimer values tended to be higher in patients with MIS-C than in those with severe COVID-19. sC5b-9 plasma levels were also significantly higher in SARS-CoV-2 infected children with acute kidney injury than without. Consistent with these findings, sC5b-9 was positively associated with creatinine and BUN, and was negatively associated with GFR, indicating that sC5b-9 is correlated with renal dysfunction (Diorio et al, Blood Advances, 2020). Therefore, complement activation plays a role in endothelial damage, thrombosis and multiorgan failure in both adults and children with COVID-19, which may have short- or long-term vascular consequences. Deregulated complement activation leads to multi-organ failure by first, fueling cytokine-driven hyper-inflammation; and second, thrombotic microangiopathy and NET-driven immunothrombosis leading to a hypercoagulable-prothrombotic state (Holter et al, PNAS, 2020). Preliminary studies of the C5-targeting monoclonal antibody, eculizumab, indicate that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation eliciting a robust anti-inflammatory and anti-thrombotic responses as reflected in a steep decline in C-reactive protein and interleukin (IL)-6 levels; reduction in NETosis; marked lung function improvement with resolution of ARDS; and marked reduction in both D-dimer levels and TAT complexes (Mastellos et al, Clincial Immunology, 2020). Therefore, complement activation is a therapeutic target in COVID-19.
Complement activation is a known driver of IL-1α production, which is high in COVID-19 (Liu et al, National Science Review, 2020). Membrane attack complex (MAC) C5b-9 attacks endothelial cells and induces synthesis of IL-1α (Bustos et al, JCI, 1997). Exposure of porcine endothelial cells to human serum containing xenoreactive antibodies and complement led to expression of IL-1α mRNA, but not IL-1β mRNA (Bustos et al, JCI, 1997). Most importantly, IL-1α is also released from necrotic and pyroptotic cells including pneumocytes and endothelial cells, the primary site of attack by SARS-CoV-2 (England et al, JBC, 2014). The effect of complement activation on IL-1α and IL-1β, and TxA2 axes has been studied by treating porcine endothelial cells with human plasma containing xenoreactive antibodies and complement (Bustos et al, JCI, 1997). In this model, there is markedly increased expression of IL-1α, COX-2 and thromboxane synthase leading to generation of TxA2. The role of IL-1α in mediating the effect of complement activation was confirmed by addition of IL-1 receptor antagonist to the human serum which prevented the release of PGE2 and TxA2 (Bustos et al, JCI, 1997). Furthermore, treatment of endothelial cells directly with IL-1α also induced expression of COX-2 mRNA while inhibition of COX-2 by an antagonist decreased the production of TxA2 by about 50% (Bustos et al, JCI, 1997). Most notably and as expected, expression of COX-1, a constitutive enzyme, was not effected by complement activation or IL-1α in this model. Therefore, complement activation and cellular necrosis may mediate thrombosis via IL-1α upregulation leading to increased expression of COX-2 and thromboxane synthase with downstream generation of TxA2 as the effector molecule for immunothrombosis in COVID-19.
Immunothrombosis in severe COVID-19 mediated by dysregulation of complement pathway, IL-1 alpha and COX-2/ thromboxane. SARS-CoV-2 induced respiratory and vascular injury leads to complement activation and inflammation. Membrane attack complex (MAC) stimulates IL-1α which is also released from dying pneumocytes, leucocytes and endothelial cells. IL-1α stimulates expression of COX-2 in endothelial cells and megakaryocytes leading to marked increase in thromboxane A2 production by endothelial cells and platelets. Thromboxane A2 initiates platelet activation, platelet-leukocyte adhesion, endothelialitis, NETosis and activation of coagulation cascade leading to microvascular thrombosis.
Depending on the infectious agent, Th0 cells polarize the immune response into T-helper type 1 (Th1), the default response in immunocompetent subjects to intracellular or phagocytosable pathogens (e.g. viruses, bacteria, protozoa, fungi) and mediated by macrophages and T- cytotoxic cells (cell-mediated immunity), or into T- helper type 2 (Th2), classically directed against extracellular nonphagocytosable pathogens, for instance helminths, and whose main effectors are eosinophils, basophils and mastocytes, as well as B cells (humoral immunity).
SARS-CoV-2 virus suppresses the host innate immune response by suppressing production IFN-lambda by epithelial, endothelial and myeloid cells (Blanco-Melo et al, Cell, 2020; Broggi et al, Science, 2020). Respiratory viruses stimulate PGD2 production by barrier epithelial cells, dendritic cells, Th2 cells, mast cells, eosinophils and macrophages (Werder et al, Sci Transl Med, 2018). PGD2 inhibits IFN-lambda expression via the DP2 receptor (Werder et al, Sci Transl Med, 2018). PGD2/CRTH2 (DP2) receptor antagonism stimulates IFN-lambda expression and limits virus replication and viral load thereby prolonging survival (Werder et al, Sci Transl Med, 2018). In COVID-19, a PGD2 antagonist would restore IFN-lambda response, reduce viral load in the upper respiratory tract, thereby inhibiting progression of the virus down the respiratory tract and into the lungs, while reducing viral transmission to household or workplace contacts (Werder et al, Sci Transl Med, 2018). This MOA of Ramatroban makes for potential efficacy in all phases of the disease including incubation, prodromal and clinical.
Maladaptive Type 2 Immune Response and Lymphopenia
SARS-CoV-2 virus induces polarization of T helper cells to a type 2 immune response, generally a hallmark of helminthic infection or allergic inflammation. Type 2 immune response is mediated by PGD2/DP2 mediated stimulation of ILC2 cells which express IL-4, IL-5 and IL-13; (Lucas et al, Nature, 2020; Gupta and Chiang, Medical Hypothesis, 2020; Trabanelli et al, Nat Commun, 2017) IL-13 stimulates proliferation of MDSC cells which suppress the cell mediated immune response and induce lymphopenia; (Trabanelli et al, Nat Commun, 2017) and IL-5 stimulates eosinophilia. (Lucas et al, Nature, 2020; Gupta and Chiang, Medical Hypothesis, 2020) Plasma PGD2 levels are markedly elevated in hospitalized COVID-19 patients (Prof. S. Reddy, UCLA, personal communication). Plasma PGD2 levels are known to increase with aging and obesity. Therefore, it is notable that lymphopenia is more severe and COVID-19 disease in more severe in the elderly.
Comorbidities in COVID-19
Old age and obesity are now recognized as the two strongest independent predictors for hospitalization in COVID-19 such that obesity can shift severe disease to younger age group. Both old age and obesity are associated with increase in generation of PGD2 and thromboxane A2 via COX-2. Therefore, ramatroban is expected to be especially effective in the obese and the elderly.
Adiposity and aging lead to increased generation of PGD2 and thromboxane A2, the two most important comorbidities associated with severity of COVID-19. Therefore, ramatroban is likely to be especially effective for prophylaxis and treatment of COVID-19 in the obese and the elderly.
Ramatroban, a potent PGD2 and thromboxane A2 antagonist is a most promising antithrombotic and immunomodulator agent for prophylaxis and treatment of SARS-CoV-2 infection and COVID-19 disease. Ramatroban is expected to reduce viral burden in the upper respiratory tract and viral transmission to contacts. PGD2 and thromboxane A2 axes are markedly stimulated with aging and obesity, and therefore, ramatroban targets the increased morbidity and mortality with COVID-19 in the obese and elderly.
Proposed mechanism of action of Ramatroban as an immunomodulator and antithrombotic agent for prophylaxis and treatment of COVID-19