Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 June 2021 doi:10.20944/preprints202106.0125.v1 On the consequences of cell fusion in COVID-19 Yuri Lazebnik1 1Lerna Consulting, New Haven, Connecticut, USA Abstract This commentary proposes how the coronavirus SARS-CoV-2 can contribute to clinical manifestations of COVID-19s by producing syncytia. Keywords: cell fusion, syncytia, hybrids, viruses, coronaviruses, SARS-CoV-2, COVID-19, blood coagulation cascade, thrombosis, cancer Corresponding author: Yuri Lazebnik Lerna Consulting 853 Orange St. New Haven, CT 06511 USA [email protected] © 2021 by the author(s). Distributed under a Creative Commons CC BY license. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 June 2021 doi:10.20944/preprints202106.0125.v1 A hallmark of severe COVID-19 is the abundance of syncytia, the products of fusion between two or Terminology and abbreviations more cells, in the lungs of the patients (Buchrieser Cell fusion: a process of merging two or more cells into et al., 2020; Bussani et al., 2020; Braga et al., one by merging their plasma membranes. 2021). These syncytia were attributed to the ability Fusogen: an agent, often a protein such as SARS-CoV-2 of spike, a protein encoded by SARS-CoV-2, to spike, capable of fusing cellular membranes. Viral fuso- fuse cells to each other, and prompted a search for gens fuse the viral envelope to the plasma membrane of the target cell and can fuse plasma membranes of adja- already approved drugs that prevent this fusion. In cent cells to each other. a recent report (Braga et al., 2021), some of such Syncytium (plural syncytia): a multinucleated cell pro- were found to inhibit TMEM16F, a protein that has duced by the fusion of two or more cells. The term comes two activities: a calcium-activated ion channel that from Greek syn "together" and kytos "box, or cell". regulates chloride secretion, and a scramblase, a Heterokaryon: a syncytium produced from more than one lipid transporter that relocates phosphatidylserine cell type, say, a pneumocyte fused to an epithelial progen- (PS) to the cell surface (Scudieri et al., 2015) in a itor or a leukocyte. process known as PS externalization. Homokaryon: a syncytium produced from cells of the PS externalization is required for cell fusion in same type, as would be the case with the fusion of two or many systems (Brukman et al., 2019; Zhang et al., more pneumocytes. 2020), which has explained why inhibiting a Cell hybrid: Mononuclear offspring of syncytia, produced scramblase prevented the formation of spike- once a syncytium undergoes mitosis. For example, hy- bridomas are made by fusing leukocytes with plasmacy- induced syncytia. However, the report has con- toma cells to obtain hybrids that produce monoclonal anti- cluded that although PS externalization “is re- bodies. quired for plasma membrane fusion, chloride se- PS (phosphatidylserine): the most abundant anionic cretion might have relevance in COVID-19 patho- (negatively charged) membrane lipid. In live cells, PS is genesis” (Braga et al., 2021). This assumption, actively moved to the cytoplasmic side of plasma mem- that the scramblase activity merely helped to iden- brane. tify the ion channel activity as a potential therapeu- Scramblases: Proteins, such as TMEM16F, that random- tic target, reflects a common opinion that syncytia ize, or scramble, the asymmetric distribution of PS across made in the body by infectious viruses are incon- the membrane, a process known as PS externalization. sequential. To evaluate this assumption let us consider how 2019) (Fig. 1). In agreement with this role, a defi- syncytia and cell fusion that creates them might be ciency of TMEM16F, the scramblase induced by involved in COVID-19. spike, is responsible for Scott syndrome, a bleed- ing disorder (Suzuki et al., 2010).Viruses and the coagulation cascade. Viral infections activate the Cell fusion as a cause of conditions associated coagulation cascade primarily by triggering the as- with the activation of the blood coagulation sembly of extrinsic tenase, a complex between cascade Tissue Factor (TF) and Factor VIIa, on the outer COVID-19 is associated with widespread throm- surface of the cellular membrane (Mackman et al., bosis (Hanff et al., 2020). Discovering syncytia in 2020) (Fig. 1). Full activation of the tenase re- COVID-19 patients led to a suggestion that “the quires PS externalization (Antoniak and Mackman, fusogenic properties of the MERS-CoV- and 2014), which, in synergy with TF binding, increas- SARS-CoV-2-infected cells might be linked to the es Factor VIIa activity by eight orders of magnitude pathogenesis of thrombosis” (Bussani et al., 2020). (Bom and Bertina, 1990; Smith, Travers and Mor- rissey, 2015). What could this link be? Since FVIIa and its precursor FVII are always pre- I would like to suggest as a candidate the scram- sent in the blood, the limiting steps for fully activat- blase activity induced by spike-mediated fusion ing FVIIa are PS externalization and the presence because PS externalization resulting from this ac- of TF capable of binding and activating FVIIa. Ac- tivity not only enables cell fusion but also controls cordingly, TF has been scrutinized as a potential the rate limiting steps of the blood coagulation target for COVID-19 therapy (Cañas et al., 2021). cascade (Zwaal, Comfurius and Bevers, 1998; Grover and Mackman, 2018; Smith and Morrissey, 2 Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 4 June 2021 doi:10.20944/preprints202106.0125.v1 tion is induced in TF-expressing cells is unclear (Smith and Morrissey, 2019). The hypothesis. The remarkable synergy of TF and externalized PS in activating FVIIa and the report that spike-induced syncytia externalize PS (Braga et al., 2021) together suggest a hypothesis that these syncytia can be a platform for assem- bling fully active extrinsic tenase capable of trig- gering blood coagulation (Fig. 2). Whether this activation would result in clinically significant thrombosis would depend on whether syncytia express TF, which is likely because endo- thelial cells express TF during viral infections, and because the lungs are abundant in other cell types that express this protein (Grover and Mackman, 2018), whether syncytia come in contact with blood, whether they are large or abundant enough Figure 1. An outline of the blood coagulation cascade to assemble sufficient number of tenase complex- Blood coagulation cascade is a network of proteases, their es, which can be tested using available clotting precursors, cofactors, cells, enzymes, feedbacks, and feed- assays, and the state of the coagulation system in forwards whose complexity and still unresolved questions a patient. Since even one thrombus can cause make this outline by necessity rudimentary, with the primary problems, the contribution of syncytia to COVID-19 goal to illustrate where the proteins that require binding to externalized PS (phosphatidylserine) for activation are in the could be clinically relevant. network. Fusion at a distance. SARS-CoV-2 spike, as oth- Most proteins involved in coagulation are called factors and er viral fusogens, can fuse cells by two mecha- are labeled by Roman numbers, such as Factor X or FX nisms. In the first, known as fusion from within, an (hence enzymes that process FX are tenases). For simplicity, infected cell begins to produce viral components, here the letter F is omitted. Activated factors are labeled with an a, as in FXa. Orange arrows represent proteolytic activity, including spike, which is transported to the plasma grey arrow show a transition between forms. Blue horizontal membrane and can fuse it to the membrane of an lines represent cellular membrane with the cell surface facing adjacent cell if that cell has a spike receptor. An- down. Accordingly, the pinheads of externalized PS also face other mechanism, fusion from without, is executed down. Note that most PS is actively relocated to face the cytoplasm unless the cell dies or the distribution is random- by viral particles or lipid vesicles studded with ized by lipid scramblases. spike that serve as a bridge between two cells (Gallaher and Bratt, 1972; Theuerkauf et al., 2021) As discussed in the text, the primary trigger of coagulation induced by viral infections is the extrinsic tenase (top left), (Fig. 2). In this case whether the virus is infectious which is a complex of TF (Tissue Factor) and FVIIa assem- or inactivated is irrelevant, as its ability to produce bled on externalized PS in the presence of calcium ions. This spike in the cell is not required. tenase produces FXa to activate enough thrombin for gener- ate the components of the intrinsic tenase, which increases Since SARS-Cov-2 infection induces the release of FXa production and thus thrombin to yield enough fibrin for extracellular vesicles (also called microvesicles) in making a thrombus, which is a meshwork of polymerized and patients with moderate or severe COVID-19 (Ro- cross-linked fibrin with entrapped blood cells, primarily plate- lets, that is large and stiff enough to obstruct a blood vessel. sell et al., 2021), and that SARS-Cov-2 spike can fuse cells if delivered on membrane vesicles Note that TF is encrypted, that is unable to activate FVIIa, (Theuerkauf et al., 2021), the site of thrombosis until it is de-encrypted by externalized PS (reviewed in (Grover and Mackman, 2018)). caused by syncytia can differ from that of infection. Thrombosis by death. Syncytia might also con- TF is regulated by controlling its expression, by tribute to thrombosis by mechanism unrelated to tissue factor pathway inhibitor (TFPI), and by prim- the induction of a scramblase. A syncytium upon ing TF through a process known as de-encryption. its demise would uncover a patch of the trombo- The primary candidate for the de-encrypter is ex- genic basement membrane.