065 Factor XA and factor VIIA utilize herpes simplex virus-associated tissue factor to increase infection through cellular protease activated receptor 2

2011 ◽  
Vol 27 (5) ◽  
pp. S84
Author(s):  
M.R. Sutherland ◽  
W. Ruf ◽  
E.L. Pryzdial
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Simplex Virus ◽  
Protease Activated Receptor 2 ◽  
Cellular Protease

Factor Xa and Factor VIIa Utilize Herpes Simplex Virus-Associated Tissue Factor to Increase Infection through Cellular Protease Activated Receptor 2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2130-2130 ◽  
Author(s):  
Michael R Sutherland ◽  
Wolfram Ruf ◽  
Ed L. Pryzdial
Keyword(s):  
Herpes Simplex ◽  
Tissue Factor ◽  
Research Funding ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Virus Surface ◽  
Simplex Virus ◽  
Umbilical Vein Endothelial Cells

Abstract Abstract 2130 Poster Board II-107 We have shown previously that purified herpes simplex type-1 (HSV1) can initiate coagulation on its surface because of tissue factor (TF) derived from the host cell and virus-encoded glycoprotein C (gC). Generation of thrombin by this viral surface pathway correlated to an increase in the susceptibility of cells to infection through a mechanism involving protease activated receptor (PAR) 1. Incomplete inhibition of the serum-mediated increase in infection by hirudin indicated that mechanisms in addition to thrombin are involved. To further investigate the importance of coagulation enzymes in HSV1 infection, the current study investigated the upstream activated coagulation factors X (FXa) and VII (FVIIa). Using a novel panel of viruses deficient in either TF and/or gC in cytolytic viral plaque assays, we examined the contribution of viral TF and gC in the FXa- or FVIIa- mediated infection of human umbilical vein endothelial cells. The addition of purified FXa resulted in up to 400% enhancement of infection when TF and gC were on the virus surface. Similarly, the effect of FVIIa was maximal when TF and gC were present, although only 150% increased. In agreement with previous studies, the addition of purified thrombin resulted in approximately a 500% increase in infection using the novel panel of viruses, which unlike FXa and FVIIa was not dependent on either viral TF or gC. Simultaneous addition of FXa/FVIIa or thrombin/FXa/FVIIa further enhanced infection to a maximum of 10-fold. FX alone had no effect on infection; however, in situ FXa generation due to the addition of FVIIa increased infection. The involvement of PAR2 was shown using inhibitory PAR2 specific antibodies which attenuated the effects of FXa and FVIIa. Anti-PAR1 inhibited the effect of thrombin, but had no effect on either FXa- or FVIIa-dependent infection. The importance of viral TF was demonstrated by inhibition of FXa or FVIIa-mediated plaque formation by anti-TF antibody, which had an insignificant effect on TF-deficient HSV1. Collectively, these data indicate that FXa and FVIIa together with TF on the virus surface may activate cellular PAR2 to increase HSV1 infection independent of thrombin. Disclosures: Ruf: Pfizer: Research Funding; Novo Nordisk: Research Funding.

Herpes Simplex Virus Infection of Endothelial Cells Is Enhanced by Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1926-1926
Author(s):  
Michael R. Sutherland ◽  
Harvey M. Friedman ◽  
Edward L. Pryzdial
Keyword(s):  
Endothelial Cells ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Tissue Factor ◽  
Culture Media ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Virus Surface ◽  
Simplex Virus

Abstract Herpes simplex virus type 1 (HSV1) and type 2 (HSV2) are highly prevalent and form life-long infections. While acute effects are not often critical, chronic infection has been linked to vascular disease. To better understand the underlying basis for the clinical correlations, our lab has demonstrated that HSV can initiate and sustain clot formation directly on the virus surface, bypassing the normal cellular requirements. This is facilitated by anionic phospholipid and tissue factor on HSV1 and HSV2, and the participation of at least HSV1- encoded glycoprotein C (gC) in factor VIIa-dependent factor X (FX) activation, emulating tissue factor. The evolution of the HSV surface as a procoagulant agent suggests that generation of thrombin is advantageous to the viral lifecycle. The current study therefore examines the hypothesis that thrombin enhances infection. Several purified HSV1 strains were evaluated, including the wild-type lab strain, MacIntyre, and the clinical isolate, NS. These viruses were compared to gC-deficient HSV1, ns-1, and the gC-restored strain, rns. As an example of HSV2, strain G, was also examined. ns-1 has been shown previously to generate less FXa per particle than the gC-containing viruses. To restrict in situ thrombin production, human umbilical vein endothelial cells were inoculated in serum-free culture media. Consistent with our hypothesis, standard plaque-forming assays revealed that co-incubation of the virus and cells with purified thrombin enhanced infection by up to 400%. The observed half-maximal concentration was approximately 20 nM thrombin for all viruses studied. As a demonstration of specificity, hirudin attenuated the effects of purified thrombin. The involvement of the protease activated receptor 1 (PAR1) in this mechanism was shown using the functional signaling analogue, thrombin receptor activating peptide (TRAP). In the absence of serum, TRAP resulted in increased infection comparable to that of purified thrombin. Approximately half the effect of TRAP was observed at 10uM, while a PAR4-activating peptide had no effect. Enabling the production of thrombin during the infection process, plaque-forming assays were also conducted with serum in the cell culture media. The presence of serum enhanced infectivity by as much as 20-fold. At a constant hirudin concentration of 5 U/ml, infection was attenuated by approximately 50% for the gC-deficient strain, ns-1, whereas the other viruses were only marginally or insignificantly affected, supporting a role for thrombin generation in situ and the participation of gC. Collectively, these data show that thrombin production by HSV enhances infection through a mechanism involving PAR1-mediated cell modulation.

Tissue Factor On The Herpes Simplex Virus Type 1 Surface Enhances Infection In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2332-2332
Author(s):  
Michael R Sutherland ◽  
Ayo Y Simon ◽  
Iryna Shanina ◽  
Marc S Horwitz ◽  
Wolfram Ruf ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Signaling ◽  
Tissue Factor ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Enveloped Viruses ◽  
Simplex Virus

Abstract Tissue factor (TF) is a multifunctional transmembrane receptor. As the pivotal initiator of the coagulant response to vascular damage, it accelerates factor (F) VIIa-dependent generation of FXa. However, TF also functions in FVIIa/FXa-dependent cell signaling via protease activated receptors (PARs), and has consequently been implicated in a wide variety of physiological and pathological conditions. Our previous work adds to this repertoire by revealing that TF, originating from the host cell membrane, is on the surface of enveloped viruses. Using herpes simplex virus type 1 (HSV1) as a model for enveloped viruses, we recently produced TF +/- HSV1 variants, which showed that viral TF enhances infection of cultured cells through a mechanism involving FXa/FVIIa-mediated activation of PAR-2. In the current project we extended these studies in vivo, hypothesizing that TF on the HSV1 surface would enhance infection by influencing the activation of coagulation proteases and/or their role in cell signaling. As before, HSV1 NS strain (with restored glycoprotein C) was propagated in TF- or genetically engineered TF+ human melanoma A7 cells, then purified and quantified. HSV1 TF+ or HSV1 TF- virus (5 x 105 virus plaque forming units of similar particle number) was injected slowly in 100 µL into the tail vein of female, eight-week old balb/c mice. On the third day, organs and blood were harvested. Live virus and total HSV1 genome were evaluated by plaque assays and rtPCR, respectively. Strikingly, production of live HSV1 TF+ (n=13) in the lung, heart, spinal cord, liver and brain was greater by an order of magnitude compared to HSV1 TF- (n=13), which was not detected in some tissues. The presence of TF on HSV1 furthermore increased virus titers in spleen and blood by several fold. Viral replication in organs was corroborated by rtPCR. The procoagulant and signaling functions of TF can be dissected using epitope-specific monoclonal antibodies (mAbs) selective for human TF. These mAbs only recognize HSV1 surface TF and not the endogenous mouse TF. These mAbs or an irrelevant mAb (TIB115, n=8) were administered by intraperitoneal injection 4 hours pre-inoculation (1 mg/mouse). When TF-dependent coagulation and the related effects on cell signaling due to attenuated protease activation was blocked by a potent combination of three mAbs to non-overlapping epitopes (5G9/6B4/9C3; 0.33 mg/mouse each, n=10), reduced levels of live HSV1 TF+ were seen in lung, heart, liver, brain and blood, with concordantly reduced viral genome levels measured by rtPCR. Similar results were obtained with mAb 5G9 alone (n=6), which selective blocks FXa generation. Interestingly, titers of HSV1 TF+ in blood and spleen were not affected by mAB 10H10 (n=6) that has no effect on coagulation, but is an effective direct inhibitor of TF signaling. Despite normal blood titers, mAb 10H10 (n=6) reduced virus titers in heart and brain, but not in other organs. These data demonstrate that virus surface TF is important for HSV1 infection in vivo and indicate that distinct functions of TF contribute to organ-specific tropism. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Protease-activated receptor 2 exacerbates cisplatin-induced nephrotoxicity

2019 ◽  
Vol 316 (4) ◽  
pp. F654-F659 ◽  
Author(s):  
Mari Watanabe ◽  
Yuji Oe ◽  
Emiko Sato ◽  
Akiyo Sekimoto ◽  
Hiroshi Sato ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Therapeutic Option ◽  
Factor Xa ◽  
Kidney Injury ◽  
Coagulation Cascade ◽  
Expression Levels ◽  
Cisplatin Nephrotoxicity ◽  
Protease Activated Receptor 2

Acute kidney injury (AKI) is associated with hypercoagulability. Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade activate protease-activated receptor 2 (PAR2). Previously, we have shown that PAR2-mediated inflammation aggravates kidney injury in models of diabetic kidney disease and adenine-induced renal fibrosis. However, the role of PAR2 in AKI remains unclear. To clarify the role of PAR2, we administered cisplatin, one of the most common causal factors of AKI, to wild-type and PAR2-deficient mice. The expression levels of tissue factor and PAR2 were significantly increased in the kidneys of mice that were administered cisplatin. A lack of PAR2 corrected the levels of plasma blood urea nitrogen and creatinine as well as ameliorated the acute tubular injury score in the kidney. A lack of PAR2 corrected the infiltration of neutrophils and the gene expression levels of proinflammatory cytokines/chemokines in these mouse kidneys. Similarly, apoptotic markers, such as cleaved caspase-3-positive area and Bax/Bcl2 ratio, were attenuated via PAR2 deletion. Thus, elevated PAR2 exacerbates cisplatin nephrotoxicity, and targeting PAR2 is a novel therapeutic option that aids in the treatment of patients with cisplatin-induced AKI.

Herpes simplex virus type 1-encoded glycoprotein C enhances coagulation factor VIIa activity on the virus

2004 ◽  
Vol 92 (11) ◽  
pp. 947-955 ◽  
Author(s):  
Michael Sutherland ◽  
Harvey Friedman ◽  
Edward Pryzdial
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Specific Antibody ◽  
Herpes Simplex Virus Type ◽  
Factor Viia ◽  
Virus Surface ◽  
Glycoprotein C ◽  
Simplex Virus

SummaryTissue factor (TF) is the blood coagulation initiator, whose cofactor function is required for physiological factor VIIa (FVIIa)-mediated activation of factor X (FX) to FXa. A previous study reported TF on herpes simplex virus type 1 (HSV1), but this explained only part of FVIIa-dependent FXa generation observed on the virus surface (Sutherland et al. (1997) Proc. Natl. Acad. Sci. USA. 94:13510-14). In the current study, we investigated the role of HSV1-encoded glycoprotein C (gC) in this process. Purified gC-deficient HSV1 facilitated several fold less FX activation by FVIIa than either wild type or gC-rescued strains.To confirm the implication of gC in FVIIa-dependent FX activation, purified soluble gC (sgC) enhanced FXa production in the absence of TF. sgC required FVIIa, calcium and anionic phospholipid to participate in FX activation, suggesting similarity to TF. When purified virus was combined with sgC, the sgC-dependent FXa generation was enhanced three orders of magnitude, suggesting synergy with an additional HSV1 component and explaining the relatively low activity of purified sgC compared to the viral counterpart. FX activation on gC-competent HSV1 was inhibited 20% by a gC-specific antibody, inhibited 40% by a TF-specific antibody, inhibited 65% by combining the gCand TF-specific antibodies, and nearly completely inhibited by the TF antibody alone on gC-deficient HSV-1. Cumulatively, these observations show that two pathways initiating FX activation function in parallel on the virus surface. In addition to the previously described TF-dependent pathway, HSV-1-encoded gC also enhances FXa generation, and like TF, requires FVIIa.

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

2020 ◽  
Vol 18 (6) ◽  
pp. 1370-1380 ◽  
Author(s):  
Bryan H. Lin ◽  
Michael R. Sutherland ◽  
Federico I. Rosell ◽  
James H. Morrissey ◽  
Edward L. G. Pryzdial
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Factor Viia ◽  
Coagulation Factor ◽  
Factor X ◽  
Herpes Simplex Virus 1 ◽  
Glycoprotein C ◽  
Simplex Virus

Assembly of VP26 in herpes simplex virus-1 capsid implicated by 3-D structure of recombinant capsid

1995 ◽  
Vol 53 ◽  
pp. 840-841
Author(s):  
Z. Hong Zhou ◽  
Jing He ◽  
Joanita Jakana ◽  
J. D. Tatman ◽  
Frazer J. Rixon ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
3D Structure ◽  
Structure Study ◽  
Herpes Simplex Virus 1 ◽  
Shell Proteins ◽  
Life Threatening ◽  
Infected Cells ◽  
Simplex Virus ◽  
Hsv 1

Herpes simplex virus-1 (HSV-1) is a ubiquitous virus which is implicated in diseases ranging from self-curing cold sores to life-threatening infections. The 2500 Å diameter herpes virion is composed of a glycoprotein spike containing, lipid envelope, enclosing a protein layer (the tegument) in which is embedded the capsid (which contains the dsDNA genome). The B-, and A- and C-capsids, representing different morphogenetic stages in HSV-1 infected cells, are composed of 7, and 5 structural proteins respectively. The three capsid types are organized in similar T=16 icosahedral shells with 12 pentons, 150 hexons, and 320 connecting triplexes. Our previous 3D structure study at 26 Å revealed domain features of all these structural components and suggested probable locations for the outer shell proteins, VP5, VP26, VP19c and VP23. VP5 makes up most of both pentons and hexons. VP26 appeared to bind to the VP5 subunit in hexon but not to that in penton.

Application of EM to differentiate herpes virus from pox virus in genital specimens

1994 ◽  
Vol 52 ◽  
pp. 262-263
Author(s):  
K. Rekrut ◽  
K. Schleuter
Keyword(s):  
Tissue Culture ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fresh Tissue ◽  
Culture Cells ◽  
Viral Transport ◽  
Carbon Coated ◽  
Immuno Assay ◽  
Simplex Virus ◽  
Enzyme Immuno Assay

Confirmation of herpes simplex virus (HSV) from genital lesions of obstetrical (OB) patients may affect both the management of the delivery and of the neonate.(l,2) During 1992 and 1993, 4,450 genital specimens from OB patients were submitted in viral transport media for herpes culture. The specimens were inoculated into MRC-5, Vero, and A-549 tissue culture tubes, incubated, and examined daily for 7 days for cytopathic effect (CPE). The original specimens were frozen at −70° C until final reports were issued. Culture tubes with CPE were tested by the Dupont Herpchek enzyme immuno assay (EIA) to confirm the presence of herpes simplex virus (HSV). (3,4) 170 OB patient specimens were positive by culture and confirmed by EIA.There were also 63 cultures exhibiting CPE ressembling HSV which were negative by EIA testing, which failed to pass in fresh tissue culture cells or progress to more enhanced CPE in culture. These original specimens were screened by electron microscopy after direct ultracentrifugation employing the Beckman airfuge with the EM 90 rotor on to formvar carbon-coated 300 mesh copper grids and negatively stained with 2% PTA.

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