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Edward L G Pryzdial - One of the best experts on this subject based on the ideXlab platform.
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Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
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coagulation factor viia binds to herpes simplex virus 1 encoded glycoprotein c forming a factor x enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
Michael R Sutherland - One of the best experts on this subject based on the ideXlab platform.
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Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
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coagulation factor viia binds to herpes simplex virus 1 encoded glycoprotein c forming a factor x enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
John P Sheehan - One of the best experts on this subject based on the ideXlab platform.
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Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation Via Direct Targeting of Factor IXa: Contribution of the Serpin-Independent Mechanism
Blood, 2011Co-Authors: Yang Buyue, John P SheehanAbstract:Abstract 2242 The relevance of factor IXa as a therapeutic target for heparin therapy remains incompletely defined. To evaluate the contribution of factor IXa inhibition, particularly serpin-independent inhibition of the intrinsic Tenase complex (factor IXa-factor VIIIa), the ability of heparin preparations to inhibit tissue factor (TF)-triggered thrombin generation was examined in human plasma. Thrombin generation was determined by a fluorogenic substrate assay employing Technothrombin TGA evaluation software to determine lag time, time to thrombin peak, peak thrombin concentration, and velocity index (slope). The inhibitory potency (EC50) of each heparin was determined by plotting concentration versus relative velocity index (ratio of the velocity index in presence and absence of heparin). Inhibition of thrombin generation was initially compared under factor IX-dependent (limiting TF) and independent (excess TF) conditions, respectively, by addition of either 0.2 or 4 pM TF to pooled normal human plasma containing increasing concentrations of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, or unfractionated heparin (UFH). UFH and Fondaparinux demonstrated complete or near complete inhibition with identical EC50 values at both tissue factor concentrations, suggesting that inhibition of intrinsic Tenase activity does not significantly contribute to their mechanism of action in plasma. In contrast, LMWH and ssLMWH demonstrated 2.9- and 5.1-fold lower EC50 values, respectively, in the presence of the limiting TF concentration. These results suggest that inhibition of intrinsic Tenase activity contributes to the mechanism of action for LMWH in plasma. The effect of heparins on the time course of plasma thrombin generation by Western blotting under similar conditions correlated well with results of the fluorogenic substrate assay. At their EC50 values, LMWH, ssLMWH and Fondaparinux clearly reduced prothrombin/meizothrombin consumption in plasma, while UFH primarily accelerated formation of the thrombin-antithrombin complex. The contribution of antithrombin to inhibition of thrombin generation was determined by comparison of antithrombin- and mock-depleted human plasmas. As expected, antithrombin depletion markedly increased the EC50 values for UFH (62–fold) and Fondaparinux (42-fold) to levels that are ∼3–8 fold higher than their expected therapeutic ranges, confirming an antithrombin-dependent mechanism of action. In contrast, antithrombin depletion increased the EC50 values more modestly for LMWH (9.4-fold) and ssLMWH (2-fold), with the EC50 for LMWH (∼ 0.7 U/ml) remaining within the therapeutic range. In the absence of antithrombin, LMWH demonstrated partial inhibition of plasma thrombin generation with a plateau representing ∼8–10% of the starting activity, remarkably similar to the inhibition of intrinsic Tenase activity with purified components. The molecular target for LMWH and ssLMWH was evaluated in dual factor IX/antithrombin-depleted plasma supplemented with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Based on both relative EC50 values for reduction in the velocity index and the maximal degree of inhibition, plasma supplemented with mutant factor IX demonstrated relative resistance to inhibition of thrombin generation by LMWH as follows: R233A > (K126A/R165A/K132A) > R170A > WT. Factor IX R233A demonstrated an 11.4-fold increase in the EC50 for LMWH relative to the wild type protease. The magnitude of differences between mutant factor IX proteins was reduced for ssLMWH relative to LMWH, but the rank order was similar. The effect of these mutations in human plasma recapitulates their effects on in vitro inhibition of intrinsic Tenase activity, confirming the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of thrombin generation by LMWH and ssLMWH. The ability of therapeutic concentrations of LMWH to directly target factor IXa in antithrombin-depleted plasma suggests that this mechanism may contribute to antithrombotic effects. Furthermore, chemo-enzymatic synthesis of “non-anticoagulant” LMWH preparations acting via this exosite-mediated antithrombotic mechanism may offer advantages in high-risk populations, particularly patients with underlying malignancy. Disclosures: Buyue: Biogen Idec Hemophilia: Employment.
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Fucosylated chondroitin sulfate inhibits plasma thrombin generation via targeting of the factor IXa heparin-binding exosite
Blood, 2009Co-Authors: Yang Buyue, John P SheehanAbstract:Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chondroitin sulfate with antithrombin-independent antithrombotic properties. Heparin cofactor II (HCII)-dependent and -independent mechanisms for DHG inhibition of plasma thrombin generation were evaluated. When thrombin generation was initiated with 0.2 pM tissue factor (TF), the half maximal effective concentration (EC50) for DHG inhibition was identical in mock- or HCII-depleted plasma, suggesting a serpin-independent mechanism. In the presence of excess TF, the EC50 for DHG was increased 13- to 27-fold, suggesting inhibition was dependent on intrinsic Tenase (factor IXa-factor VIIIa) components. In factor VIII–deficient plasma supplemented with 700 pM factor VIII or VIIIa, and factor IX–deficient plasma supplemented with plasma-derived factor IX or 100 pM factor IXa, the EC50 for DHG was similar. Thus, cofactor and zymogen activation did not contribute to DHG inhibition of thrombin generation. Factor IX–deficient plasma supplemented with mutant factor IX(a) proteins demonstrated resistance to DHG inhibition of thrombin generation [factor IX(a) R233A > R170A > WT] that inversely correlated with protease-heparin affinity. These results replicate the effect of these mutations with purified intrinsic Tenase components, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition by DHG. Glycosaminoglycan-mediated intrinsic Tenase inhibition is a novel antithrombotic mechanism with physiologic and therapeutic applications.
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the heparin binding exosite is critical to allosteric activation of factor ixa in the intrinsic Tenase complex the role of arginine 165 and factor x
Biochemistry, 2007Co-Authors: Tina M. Misenheimer, Yang Buyue, John P SheehanAbstract:Heparin inhibits the intrinsic Tenase complex (factor IXa−factor VIIIa) via interaction with a factor IXa exosite. To define the role of this exosite, human factor IXa with alanine substituted for conserved surface residues (R126, N129, K132, R165, N178) was characterized. Chromogenic substrate hydrolysis by the mutant proteases was reduced 20−30% relative to factor IXa wild type. Coagulant activity was moderately (N129A, K132A, K126A) or dramatically (R165A) reduced relative to factor IXa wild type. Kinetic analysis demonstrated a marked reduction in apparent cofactor affinity (23-fold) for factor IXa R165, and an inability to stabilize cofactor activity. Factor IXa K126A, N129A, and K132A demonstrated modest reductions (∼2-fold) in apparent cofactor affinity, and accelerated decay of intrinsic Tenase activity. In the absence of factor VIIIa, factor IXa N178A and R165A demonstrated a defective Vmax(app) for factor X activation. In the presence of factor VIIIa, Vmax(app) varied in proportion to the predic...
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depolymerized holothurian glycosaminoglycan and heparin inhibit the intrinsic Tenase complex by a common antithrombin independent mechanism
Blood, 2005Co-Authors: John P Sheehan, Erik N WalkeAbstract:Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties and inhibits factor X activation by the intrinsic Tenase complex (factor IXa–factor VIIIa). The mechanism and molecular target for intrinsic Tenase inhibition were determined and compared with inhibition by low-molecular-weight heparin (LMWH). DHG inhibited factor X activation in a noncompetitive manner (reduced Vmax(app)), with 50-fold higher apparent affinity than LMWH. DHG did not affect factor VIIIa half-life or chromogenic substrate cleavage by factor IXa–phospholipid but reduced the affinity of factor IXa for factor VIIIa. DHG competed factor IXa binding to immobilized LMWH with an EC50 35-fold lower than soluble LWMH. Analysis of intrinsic Tenase inhibition, employing factor IXa with mutations in the heparin-binding exosite, demonstrated that relative affinity (Ki) for DHG was as follows: wild type > K241A > H92A > R170A > > R233A, with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa–LMWH affinity and the potency of LMWH for intrinsic Tenase. DHG also accelerated decay of the intact intrinsic Tenase complex. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa–factor VIIIa interactions.
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heparin inhibits the intrinsic Tenase complex by interacting with an exosite on factor ixa
Biochemistry, 2003Co-Authors: John P Sheehan, Catherine E Kobbervig, Heidi M KirkpatrickAbstract:The specific molecular target for direct heparin inhibition of factor X activation by intrinsic Tenase (factor IXa−factor VIIIa) was investigated. Comparison of size-fractionated oligosaccharides demonstrated that an octasaccharide was sufficient to inhibit intrinsic Tenase. Substitution of soluble dihexanoic phosphatidylserine (C6PS) for phospholipid (PL) vesicles demonstrated that inhibition by low-molecular weight heparin (LMWH) was independent of factor IXa−factor VIIIa membrane assembly. LMWH also inhibited factor X activation by the factor IXa−PL complex via a distinct mechanism that required longer oligosaccharides and was independent of substrate concentrations. The apparent affinity of LMWH for the factor IXa−PL complex was higher in the absence of factor VIIIa, suggesting that the cofactor adversely affected the interaction of heparin with factor IXa-phospholipid. LMWH did not interact directly with the active site, as it failed to inhibit chromogenic substrate cleavage by the factor IXa−PL comp...
Anastasia N Sveshnikova - One of the best experts on this subject based on the ideXlab platform.
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substrate delivery mechanism and the role of membrane curvature in factor x activation by extrinsic Tenase
Journal of Theoretical Biology, 2017Co-Authors: Tatiana A Kovalenko, Mikhail A Panteleev, Anastasia N SveshnikovaAbstract:Abstract Membrane-bound enzyme complex of extrinsic Tenase (VIIa/TF) is believed to be the primary activator of blood clotting in vivo . This complex (where factor VIIa (FVIIa) is a catalytically active part and tissue factor (TF) is its essential cofactor) activates its primary substrate factor X (FX) leading to factor Xa (FXa) (‘a’ stands for ‘activated’). Both FX and FXa are able to bind to phospholipid membrane and, therefore, are distributed between solution and membrane surface. As a result, two possible mechanisms of substrate delivery to the extrinsic Tenase exist: via lateral diffusion on the membrane surface or directly from the solution. Determination of the predominant pathway of substrate delivery is an important key to understanding the precise reaction mechanism. Here we construct a mechanism-driven computational model of FX activation by extrinsic Tenase on the surface of phospholipid vesicles of different size. We show that experimentally observed dependence of the Tenase activity on the phospholipid concentration could be obtained only if the substrate (FX) is membrane-bound. For correct experimental data description it is also necessary to take into account the dependence of FX/FXa membrane binding parameters (equilibrium dissociation constant and the number of phospholipid molecules per bound FX/FXa) on the membrane curvature. The model predicts that small vesicles promote activation of FX by the extrinsic Tenase significantly better than large vesicles (with the same overall phospholipid, factors VIIa, X and TF concentrations in the solution).
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the mechanisms and kinetics of initiation of blood coagulation by the extrinsic Tenase complex
Biophysics, 2017Co-Authors: Mikhail A Panteleev, T A Kovalenko, Anastasia N SveshnikovaAbstract:The system of hemostasis includes coagulation of blood plasma and formation of platelet aggregate. Plasma clotting is a cascade of proteolytic reactions, triggered by the contact of blood plasma with any tissue except the normal vessel endothelium. During the contact an enzymatic complex is formed of the soluble blood plasma protein, factor VIIa, and a membrane-anchored protein, tissue factor. This complex is called extrinsic Tenase; it is the key initiator of blood coagulation. The main substrates of extrinsic Tenase are blood plasma factors X and IX. During the reaction they undergo proteolytic cleavage and become active serine proteases, factors Xa and IXa, respectively. Factor Xa in complex with its cofactor factor Va catalyzes formation of the key coagulation enzyme, thrombin, which leads to fibrin polymerization and plasma gelation. Although all of the proteins that participate in this process have been known for a long time, several questions remain unanswered. As an example, what is the role of the reaction surface on which the complex is formed, what is the role of membrane-bound multimeres of factor X (Xa), and in what way does the activation of the factor VII proceed? Here, we review recent theoretical and experimental works focused on the biophysical mechanisms of extrinsic Tenase functioning and discuss some of these problems.
James H Morrissey - One of the best experts on this subject based on the ideXlab platform.
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Coagulation factor VIIa binds to herpes simplex virus 1‐encoded glycoprotein C forming a factor X‐enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
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coagulation factor viia binds to herpes simplex virus 1 encoded glycoprotein c forming a factor x enhanced Tenase complex oriented on membranes
Journal of Thrombosis and Haemostasis, 2020Co-Authors: Michael R Sutherland, Federico I Rosell, James H Morrissey, Edward L G PryzdialAbstract: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.
