Thrombin A-Chain: Activation Remnant or Allosteric Effector?

Although prothrombin is one of the most widely studied enzymes in biology, the role of the thrombin A-chain has been neglected in comparison to the other domains. This paper summarizes the current data on the prothrombin catalytic domain A-chain region and the subsequent thrombin A-chain. Attention is given to biochemical characterization of naturally occurring prothrombin A-chain mutations and alanine scanning mutants in this region. While originally considered to be simply an activation remnant with little physiologic function, the thrombin A-chain is now thought to play a role as an allosteric effector in enzymatic reactions and may also be a structural scaffold to stabilize the protease domain

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

BackgroundThe 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.ObjectiveTo examine the biochemical influences of gC on FVIIa‐dependent FX activation.MethodsImmunogold 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.ResultsIEM 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.ConclusionsThe 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.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155933/1/jth14790-sup-0001-Supinfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155933/2/jth14790.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155933/3/jth14790_am.pd

A biochemical network can control formation of a synthetic material by sensing numerous specific stimuli

Developing bio-compatible smart materials that assemble in response to environmental cues requires strategies that can discriminate multiple specific stimuli in a complex milieu. Synthetic materials have yet to achieve this level of sensitivity, which would emulate the highly evolved and tailored reaction networks of complex biological systems. Here we show that the output of a naturally occurring network can be replaced with a synthetic material. Exploiting the blood coagulation system as an exquisite biological sensor, the fibrin clot end-product was replaced with a synthetic material under the biological control of a precisely regulated cross-linking enzyme. The functions of the coagulation network remained intact when the material was incorporated. Clot-like polymerization was induced in indirect response to distinct small molecules, phospholipids, enzymes, cells, viruses, an inorganic solid, a polyphenol, a polysaccharide, and a membrane protein. This strategy demonstrates for the first time that an existing stimulus-responsive biological network can be used to control the formation of a synthetic material by diverse classes of physiological triggers

Coagulation factor Va Glu-96-Asp-111: a chelator-sensitive site involved in function and subunit association.

Coagulation FVa (factor Va) accelerates the essential generation of thrombin by FXa (factor Xa). Although the noncovalent Ca2+-dependent association between the FVa light and heavy subunits (FVaL and FVaH) is required for function, little is known about the specific residues involved. Previous fragmentation studies and homology modelling led us to investigate the contribution of Leu-94-Asp-112. Including prospective divalent cation-binding acidic amino acids, nine conserved residues were individually replaced with Ala in the recombinant B-domainless FVa precursor (DeltaFV). While mutation of Thr-104, Glu-108, Asp-112 or Tyr-100 resulted in only minor changes to FXa-mediated thrombin generation, the functions of E96A (81%), D111A (70%) and D102A (60%) mutants (where the single-letter amino acid code is used) were notably reduced. The mutants targeting neighbouring acidic residues, Asp-79 and Glu-119, had activity comparable with DeltaFV, supporting the specific involvement of select residues. Providing a basis for reduced activity, thrombin treatment of D111A resulted in spontaneous dissociation of subunits. Since FVaH and FVaL derived from E96A or D102A remained associated in the presence of Ca2+, like the wild type, but conversely dissociated rapidly upon chelation, a subtle difference in divalent cation co-ordination is implied. Subunit interactions for all other single-point mutants resembled the wild type. These data, along with corroborating multipoint mutants, reveal Asp-111 as essential for FVa subunit association. Although Glu-96 and Asp-102 can be mutated without gross changes to divalent cation-dependent FVaH-FVaL interactions, they too are required for optimal function. Thus Glu-96-Asp-111 imparts at least two discernible effects on FVa coagulation activity

Ca2+-dependent and phospholipid-independent binding of annexin 2 and annexin 5.

Annexins are a family of homologous proteins that associate with anionic phospholipid (aPL) in the presence of Ca(2+). Evidence that the function of one annexin type may be regulated by another was recently reported in studies investigating cytomegalovirus-aPL interactions, where the fusogenic function of annexin 2 (A2) was attenuated by annexin 5 (A5). This observation suggested that A2 may bind directly to A5. In the present study, we demonstrated this interaction. The A2-A5 complex was first detected utilizing (covalently linked) fluorescein-labelled A5 (F-A5) as a reporter group. The interaction required concentrations of Ca(2+) in the millimolar range, had an apparent dissociation constant [ K (d)(app)] of 1 nM at 2 mM Ca(2+) and was independent of aPL. A2 bound comparably with F-A5 pre-equilibrated with an amount of aPL that could bind just the F-A5 or to an excess amount of aPL providing sufficient binding sites for all of F-A5 and A2. A2-A5 complex formation was corroborated in an experiment, where [(125)I]A2 associated in a Ca(2+)-dependent manner with A5 coated on to polystyrene. Surface plasmon resonance was used as a third independent method to demonstrate the binding of A2 and A5 and, furthermore, supported the conclusion that the monomeric and tetrameric forms of A2 bind equivalently to A5. Together these results demonstrate an A2-A5 interaction and provide an explanation as to how A5 inhibits the previously reported A2-dependent enhancement of virus-aPL fusion

Herpes simplex virus type 1-encoded glycoprotein C contributes to direct coagulation Factor X–virus binding

The HSV1 (herpes simplex virus type 1) surface has been shown recently to initiate blood coagulation by FVIIa (activated Factor VII)-dependent proteolytic activation of FX (Factor X). At least two types of direct FX–HSV1 interactions were suggested by observing that host cell-encoded tissue factor and virus-encoded gC (glycoprotein C) independently enhance FVIIa function on the virus. Using differential sedimentation to separate bound from free (125)I-ligand, we report in the present study that, in the presence of Ca(2+), FX binds directly to purified wild-type HSV1 with an apparent dissociation constant (K(d)) of 1.5±0.4 μM and 206±24 sites per virus at saturation. The number of FX-binding sites on gC-deficient virus was reduced to 43±5, and the remaining binding had a lower K(d) (0.7±0.2 μM), demonstrating an involvement of gC. Engineering gC back into the deficient strain or addition of a truncated soluble recombinant form of gC (sgC), increased the K(d) and the number of binding sites. Consistent with a gC/FX stoichiometry of approximately 1:1, 121±6 (125)I-sgC molecules were found to bind per wild-type HSV1. In the absence of Ca(2+), the number of FX-binding sites on the wild-type virus was similar to the gC-deficient strain in the presence of Ca(2+). Furthermore, in the absence of Ca(2+), direct sgC binding to HSV1 was insignificant, although sgC was observed to inhibit the FX–virus association, suggesting a Ca(2+)-independent solution-phase FX–sgC interaction. Cumulatively, these data demonstrate that gC constitutes one type of direct FX–HSV1 interaction, possibly providing a molecular basis for clinical correlations between recurrent infection and vascular pathology