Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced tenase complex oriented on membranes

Abstract

Background The cell membrane‐derived initiators of coagulation, tissue factor (TF) and anionic phospholipid (aPL), are constitutive on the herpes simplex virus type 1 (HSV1) surface, bypassing physiological regulation. TF and aPL accelerate proteolytic activation of factor (F) X to FXa by FVIIa to induce clot formation and cell signaling. Thus, infection in vivo is enhanced by virus surface TF. HSV1‐encoded glycoprotein C (gC) is implicated in this tenase activity by providing viral FX binding sites and increasing FVIIa function in solution. Objective To examine the biochemical influences of gC on FVIIa‐dependent FX activation. Methods Immunogold electron microscopy (IEM), kinetic chromogenic assays and microscale thermophoresis were used to dissect tenase biochemistry. Recombinant TF and gC were solubilized (s) by substituting the transmembrane domain with poly‐histidine, which could be orientated on synthetic unilamellar vesicles containing Ni‐chelating lipid (Ni‐aPL). These constructs were compared to purified HSV1 TF±/gC ± variants. Results IEM confirmed that gC, TF, and aPL are simultaneously expressed on a single HSV1 particle where the contribution of gC to tenase activity required the availability of viral TF. Unlike viral tenase activity, the cofactor effects of sTF and sgC on FVIIa was additive when bound to Ni‐aPL. FVIIa was found to bind to sgC and this was enhanced by FX. Orientation of sgC on a lipid membrane was critical for FVIIa‐dependent FX activation. Conclusions The assembly of gC with FVIIa/FX parallels that of TF and may involve other constituents on the HSV1 envelope with implications in virus infection and pathology.

Antiviral anticoagulation

“…106,[150][151][152] Demonstrating specificity, these activities are inhibited by direct antagonists of TF function. 150,151 These data imply the availability of envelope aPL for macromolecular assembly of the viral TF-FVIIa-FX complex (Figure 1). Consistent with these observations, the aPL-binding protein, annexin A5, labeled with a medium-diameter gold bead, binds to TF-positive HSV1 indicating a calcium-dependent interaction with the virus (Figure 2).…”

“…Using HSV1 propagated in TF-expressing cultured cells and purified by sucrose gradient differential ultracentrifugation, 74 multiple-sized electron-dense gold beads were used to simultaneously distinguish 3 constituents on a single HSV1. 150 Only when grown in TF-bearing cell types do purified HSV1 preparations initiate plasma clotting 106,150 and FVIIa-dependent FX activation in experimental systems using purified proteins. 106,[150][151][152] Demonstrating specificity, these activities are inhibited by direct antagonists of TF function.…”

“…150 Only when grown in TF-bearing cell types do purified HSV1 preparations initiate plasma clotting 106,150 and FVIIa-dependent FX activation in experimental systems using purified proteins. 106,[150][151][152] Demonstrating specificity, these activities are inhibited by direct antagonists of TF function. 150,151 These data imply the availability of envelope aPL for macromolecular assembly of the viral TF-FVIIa-FX complex (Figure 1).…”

“…Consistent with these observations, the aPL-binding protein, annexin A5, labeled with a medium-diameter gold bead, binds to TF-positive HSV1 indicating a calcium-dependent interaction with the virus (Figure 2). These data show the 2 essential cellular initiators of F I G U R E 2 Coagulation initiators tissue factor (TF) and anionic phospholipid (aPL) are available on the herpes simplex virus type 1 (HSV1) surface 150 . Representative triple-labeled immunogold electron micrographs simultaneously identifying the HSV1 marker, glycoprotein C (gC; 15 nm gold bead), aPL (10 nm bead), and TF (6 nm gold bead).…”

“…We have reported that purified gC and gC on the virus surface mimics the cofactor function of TF toward FVIIaenhanced FXa generation. 150,151 Like TF, it binds directly to both FVIIa and FX forming a cofactor-protease-substrate complex. 150 A further similarity to TF is that FX stabilizes gC-FVIIa cofactor-protease assembly, which for TF would rigorously localize the hemostatic response to sites of aPL accessibility.…”