Novel mechanism regulating tissue factor activity.

The phospholipid composition of plasma membranes and extracellular vesicles (EVs) affects coagulation in several ways. In this issue of Blood, Wang et al show that a phospholipid-degrading enzyme, acid sphingomyelinase (ASMase), translocates from lysosomes to the plasma membrane of macrophages upon infection with severe acute respiratory syndrome coronavirus 2 spike protein pseudovirus (SARS-CoV-2-SP-PV).1

Translocation of ASMase reduces membrane staining for sphingomyelin (SM), the phospholipid substrate of ASMase, confirming that the translocated enzyme remains active. Concurrently, an increase of tissue factor (TF) activity is observed, which is sensitive to pharmacological inhibition, gene silencing, and inhibition of virus entry, but insensitive to inhibition of phosphatidylserine (PS), another phospholipid involved in regulating TF activity. Earlier, the authors described a similar involvement of ASMase in lipopolysaccharide- and cytokine-induced TF activation.2

TF is the transmembrane receptor for coagulation factor VII (FVII). TF is expressed by extravascular cells under physiological conditions, and TF is present on EVs in normal human body fluids such as saliva, urine, and milk.3, 4 TF is also found in the blood, where it is expressed by monocytes and endothelial cells during infection and inflammation. TF triggers coagulation by binding FVII, thereby promoting the formation of active FVII (FVIIa). Often, however, TF does not trigger coagulation, and several posttranslational mechanisms have been described regulating the procoagulant activity of “cryptic” TF, including homodimerization, glycosylation, oxidation of disulfide bonds, and exposure of PS.5 Exposure of PS also provides a negatively charged membrane surface to which coagulation factors such as FVa can bind in the presence of calcium ions.

In resting cells, PS and other charged phospholipids are present in the inner leaflet of the phospholipid bilayer of the membrane, whereas uncharged phospholipids such as SM are present in the outer leaflet (see figure ). This phospholipid asymmetry is actively maintained by phospholipid transporters; for example, upon platelet activation, a PS-specific transporter is inhibited, resulting in the exposure of PS on platelets and EVs.6 Earlier, Del Conde et al showed that EVs bearing TF from human monocytic cells interact and fuse with activated platelets, thereby depositing TF in a PS-rich environment that promotes coagulation.7 The Wang study now provides evidence for an SM-dependent but PS-independent mechanism regulating this TF procoagulant activity.

Phospholipid-dependent mechanisms regulating TF procoagulant activity. (A) Resting cells have an asymmetric phospholipid distribution. PS is located in the inner leaflet of the membrane but translocates upon cell activation. In turn, FX binds to the membrane and is activated by the TF and FVIIa complex to FXa. (B) Upon infection with SARS-CoV-2-SP-PV, ASMase translocates from lysosomes to the plasma membrane. SM, the substrate of ASMase, is present in the outer leaflet and is hydrolyzed by SM into ceramide and phosphorylcholine, thereby triggering the TF procoagulant activity and activating FX to FXa. Professional illustration by Patrick Lane, ScEYEnce Studios.

The present study does not address the mechanism underlying translocation of ASMase. When the translocated ASMase is enzymatically active, cell lysis may occur, as described previously for bacterial sphingomyelinases,8 which may potentially give access to intracellular TF. Infection with pseudovirus is reported to result in the release of TF-exposing EVs. However, more evidence is needed as nanoparticle-tracking analysis detects all particles above the detection limit in suspension, not just EVs, and conventional flow cytometry is too insensitive to detect single EVs with a diameter of 150 nm and smaller.9

There is ample evidence that SM is attacked by sphingomyelinases secreted by pathogenic bacteria.8 To which extent lysis of SM by intracellular (eg, virus induced) and extracellular (eg, bacteria-secreted) sphingomyelinases contributes to decryption (ie, activation) of TF and thrombosis in pathological conditions requires further investigation. Recently, Lacroix and coworkers reported that the TF activity of EVs in patients with severe COVID-19 is strongly increased compared with patients with septic shock, and this increased TF activity is associated with an increased thrombotic risk.10

Whether ASMase played a role in the decryption of TF activity observed by Lacroix and coworkers is unknown, but investigating the presence of bacterial (extracellular) sphingomyelinases and determining the lipid composition of EVs in patient blood may provide evidence for involvement of intracellular and extracellular sphingomyelinases in decryption of TF activity in vivo. If proven, this may offer new therapeutic targets against thrombosis.