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Role of herpes simplex virus type-1-encoded glycoprotein C in coagulation factor X binding to the virus Livingston, Joel Reed

Abstract

Members of the Herpesvirus family, including herpes simplex virus type 1 (HSV-1) have been linked to vascular disease due to their ability to promote a procoagulant phenotype within the host vasculature. Previous studies have shown that HSV-1 is able to activate the blood coagulation zymogen, factor X (FX) to factor Xa (FXa). FXa, the enzymatic portion of the prothombinase complex, cleaves prothrombin into thrombin, which causes the generation of the fibrin clot and also functions as a potent cell agonist. The production of FXa is normally limited to the site of vascular damage, where procoagulant, anionic phospholipid (aPL) and tissue factor (TF) become exposed and available to plasma clotting factors. Exposure of aPL allows FX to bind to the phospholipid membrane in a Ca²⁺-dependent manner, whereas TF exposure leads to the formation of a TF-factor Vila (FVIIa) complex, known as tenase, which activates FX to FXa. Previously, our laboratory has reported that HSV-1 possesses aPL and TF, enabling them to activate FX and subsequently generate thrombin prior to affecting the host cell. It was also shown that a TF-independent pathway for FX activation involving the virus-encoded protein, glycoprotein C (gC), exists on the HSV-1 surface. Using differential sedimentation to separate bound from free 125l-ligand, we now report that in the presence of Ca²⁺, FX binds directly to purified wild type HSV-1 with an apparent dissociation constant (Kd) of 1.5 μM and has approximately 200 binding sites per virus. Demonstrating an involvement of gC, the number of FX binding sites on gC-deficient virus was reduced to 43, and residual binding had an altered KD ( 0.7 | JM ). Engineering gC back into the deficient strain or addition of a soluble recombinant form of gC (sgC), returned binding to that of wild type. Consistent with a gC:FX stoichiometry of 1:1, approximately 100 ¹²⁵l - sgC were found to bind per virus. In the absence of Ca²⁺, the number of FX binding sites on the wild type virus was similar to the gC-deficient strain in the presence of Ca²⁺ , and direct sgC-binding was insignificant. Under these conditions, sgC inhibited the FX-virus association, suggesting a Ca²⁺-independent solution-phase interaction. Providing a molecular basis for clinical correlations between recurrent infection and vascular pathology, these data demonstrate that gC constitutes one type of direct FX-HSV-1 interaction with the likely configuration FX-gC-Ca²⁺ - virus .

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