Thrombosis

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Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. When a blood vessel is injured, the body uses platelets (thrombocytes) and fibrin to form a blood clot to prevent blood loss. Thrombosis can lead to stroke, myocardial infarction, limb ischemia, etc. Different types of thrombotic disorders include disseminated intravascular coagulation, deep venous thrombosis, venous thromboembolism, arterial thrombosis, pulmonary embolism, etc. Thrombosis can play a part in the pathogenesis of many conditions including viral and bacteria infections, and hematological disorders. Ramatroban is a promising treatment for thrombosis.

Thromboxane A2/TP Receptor

Antagonism

Anti-Platelet Agent

Ramatroban is a potential antagonist of the thromboxane prostanoid (TP) receptor. The activation of the TP receptor on platelets, monocytes/macrophages, endothelial cells and vascular smooth muscle cells plays an important role in regulating platelet activation and vascular tone and in the pathogenesis of thrombosis and vascular inflammation. Oxidative stress and vascular inflammation increase the formation of TP receptor agonists, which promote initiation and progression of atherogenesis and thrombosis. Furthermore, TP receptor activation promotes angiogenesis and vessel wall constriction. Besides thromboxane A2 and its endoperoxide precursors, prostaglandin G2and H2, isoprostanes, and 20-hydroxyeicosatetraenoic acid also activate TP receptor as autocrine or paracrine ligands. These additional TP activators play a role in pathological conditions such as diabetes, obesity, and hypertension, and their biosynthesis is not inhibited by aspirin, at variance with that of thromboxane A2 (Capra et al, 2014). Therefore, ramatroban will not only inhibit the effects of thromboxane A2, but also other TP receptor agonists. 

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); and third, ramatroban should be effective in the elderly and the obese since it blocks both COX-1 and COX-2 mediated thromboxane A2, while low-dose aspirin only inhibits COX-1.

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Prevents Formation of Neutrophil Extracellular Traps (NETs)
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Volker Brinkmann

TP receptor activation on platelets leads to P-selectin expression, 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, including COVID-19. 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). It is noteworthy that tissue factor is expressed on NETs (Kambas et al, 2013), which may further exacerbate vasculitis and thrombosis. 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)

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Abrogates CLEC2 Signaling
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(Sung and Hsieh, Frontiers in Immunology, 2019)

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. 

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(Badolia et al, JCB, 2017)

Prostaglandin D2/DP2 Receptor

Antagonism

Interestingly, a stable metabolite of thromboxane A2, 11-dehydro-thromboxane B2 is a full agonist of the PGD2/DP2 receptor (Bohm et al, 2004). Ramatroban is also a potential antagonist of the DP2 receptor. The DP2 receptor is mainly expressed by the key cells involved in type 2 immune responses, including TH2 cells, type 2 innate lymphoid cells and eosinophils. The DP2 receptor pathway is a novel and important therapeutic target for asthma, because increased prostaglandin D2 (PGD2) production induces significant inflammatory cell chemotaxis and degranulation via its interaction with the DP2 receptor. PGD2 is a lipid mediator, predominantly released from mast cells, but also by other immune cells such as TH2 cells and dendritic cells, which plays a significant role in the pathophysiology of asthma. PGD2 mainly exerts its biological functions via two G-protein-coupled receptors, the PGD2 receptor 1 (DP1) and 2 (DP2) (Domingo et al, 2018). 

PGD2 is also produced by platelets which further stimulates platelets in an autocrine manner (Cooper and Ahern, 1979). DP2 agonism is involved in platelet activation while DP1 abrogates platelet activation (Braune et al, 2020). DP1 is coupled with a Gsα protein which increases the concentration of intracellular cAMP (Boie et al, 1995). In contrast, CRTH2 is genetically closer to chemotactic receptors, such as chemokine receptors and the leukotriene B4 receptor, coupled with Giα protein, which causes an increase in calcium and a decrease in concentrations of cAMP (Oguma et al, 2008). Therefore, blocking the DP2 receptor with ramatroban may not only inhibit DP2 mediated platelet activation, but also increased bioavailability of PGD2 for the DP1 receptor to further inhibit platelet activation and aggregation.