SARS-CoV-2 Spike protein activates TMEM16F-mediated platelet procoagulant activity

Abstract

Thrombosis of the lung microvasculature is a characteristic of COVID-19 disease, which is observed in large excess compared to other forms of acute respiratory distress syndrome and thus suggests a trigger for thrombosis that is endogenous to the lung. Our recent work has shown that the SARS-CoV-2 Spike protein activates the cellular TMEM16F chloride channel and scramblase. Through a screening on >3,000 FDA/EMA approved drugs, we identified Niclosamide and Clofazimine as the most effective molecules at inhibiting Spike-induced TMEM16 activation. As TMEM16F plays an important role in stimulating the procoagulant activity of platelets, we investigated whether Spike directly affects platelet activation and pro-thrombotic function and tested the effect of Niclosamide and Clofazimine on these processes. Here we show that Spike, present either on the virion envelope or on the cell plasma membrane, promotes platelet activation, adhesion and spreading. Spike was active as a sole agonist or, even more effectively, by enhancing the function of known platelet activators. In particular, Spike-induced a marked procoagulant phenotype in platelets, by enhancing Ca2+ flux, phosphatidylserine externalization on the platelet outer cell membrane, and thrombin generation. Eventually, this increased thrombin-induced clot formation and retraction. Both Niclosamide and Clofazimine blocked this Spike-induced procoagulant response. These findings provide a pathogenic mechanism to explain lung thrombosis-associated with severe COVID-19 infection. We propose that Spike, present in SARS-CoV-2 virions or exposed on the surface of infected cells in the lungs, enhances the effects of inflammation and leads to local platelet stimulation and subsequent activation of the coagulation cascade. As platelet TMEM16F is central in this process, these findings reinforce the rationale of repurposing Niclosamide for COVID-19 therapy.

Circulating Reelin promotes inflammation and modulates disease activity in acute and long COVID-19 cases

“…Thereby, Reelin promotes platelet activation leading to thrombin generation and the formation of a fibrin clot. Since Reelin greatly increases with the severity of the infection, its prothrombotic function may participate in or aggravate the thromboembolic complications seen in COVID-19 patients ( 1 – 3 ), and may reflect an extensive platelet activation as seen in the severe forms ( 42 ) ( Figure 4 ). We have shown previously that Reelin promotes vascular adhesion and permeability to leukocytes, and in this article that Reelin expression in plasma is greatly elevated by ongoing inflammation anywhere in the body.…”

The Dual-Targeted Fusion Inhibitor Clofazimine Binds to the S2 Segment of the SARS-CoV-2 Spike Protein

“…As Clofazimine has demonstrated synergistic antiviral activity with other direct-acting antivirals, such as Remdesivir [ 5 ], we hope that fusion inhibitors with this mechanism of action may be considered for future synergistic drug combination therapies [ 15 ]. More recent mechanistic studies have also shown that Clofazimine is able to inhibit Spike-induced activation of TMEM16 and subsequent procoagulant activity [ 57 ]. This observation may increase clinical interest in using Clofazimine as an experimental drug in the treatment of COVID-19 infections with significant pulmonary thrombosis, or in the treatment of a range of other Spike-induced pathologies, potentially including the treatment of “long COVID” [ 58 , 59 , 60 , 61 , 62 , 63 , 64 ].…”

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract

“…Moreover, enhancing endosomal pH and lowering intracellular Ca 2+ levels by niclosamide may both reduce viral uptake [ 23 , 25 , 84 ]. Finally, niclosamide inhibits the formation of pneumocyte syncytia and thrombus formation induced by the spike protein of SARS-CoV-2, which is due to the inhibition of ANO6 and possibly ANO1 [ 5 , 9 , 18 , 71 ]. Taken together, these findings support further evaluation of niclosamide for the use in inflammatory airway diseases.…”

“…In contrast, ivermectin reduces viral infection by inhibiting importins, soluble transport receptors that are essential for the nucleo-cytoplasmic transit of substrates [ 17 , 51 , 59 ]. Niclosamide and ivermectin are suggested to inhibit ANO1 currents and ANO6-mediated phospholipid scrambling that is crucial for formation of multinucleated airway epithelial cells (syncytia) and blood clotting observed during SARS-CoV-2 infection and after vaccination against COVID-19 [ 9 , 18 , 71 ].…”