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

Structure of complement C3(H2O) revealed by quantitative cross-linking/mass spectrometry and modelling:QCLMS and modelling reveals structure of C3(H2O)

The slow but spontaneous and ubiquitous formation of C3(H(2)O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H(2)O) has not been determined. Here we subjected C3(H(2)O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H(2)O) and C3, as well as between C3(H(2)O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H(2)O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H(2)O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H(2)O) domain architecture. The unique arrangement of domains thus observed in C3(H(2)O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H(2)O) and C3b in terms of complement activation and regulation

Maestro tissue factor reaches new hEIGHT

In this issue of Blood, Kamikubo et al broaden the already diverse tissue factor (TF) repertoire to include activation of anti–hemophilia A clotting factor VIII (FVIII), which amplifies coagulation initiation.1</jats:p

Understanding help-seeking in rural men of the Southern Interior : a qualitative study

Background: Men disproportionately experience the leading causes of death (e.g., heart disease, cancer), but are often hesitant to seek services for their health concerns. Additionally, suicide rates are higher for men, but they are less likely to be diagnosed with depression. Men’s under-reported illness and lower healthcare utilization is important in rural communities, where there are higher populations of older residents, less available local resources and less access to transportation to health services and that may further limit men’s intentions to seek help. This study aimed to gain an understanding of men’s perceptions and behaviours surrounding help-seeking. Research Design: Interpretive descriptive qualitative methods were used for the study. Data were collected via interviews with men living in two rural regions of the Southern Interior of British Columbia. Informal Community Conversations were also conducted in each region. Findings: Four themes on help-seeking were identified: environmental barriers (e.g., ruralness of communities, lack of health services); lack of health education or awareness; masculinity, with both positive and negative impacts; and informal support as a facilitator to help-seeking. Discussion: Men living in the rural communities of the Southern Interior face a range of external and internal barriers that limit their capacity to seek help when needed. Themes informed the thinking around a future framework of rural men’s help-seeking that highlighted the duality of these barriers. Additionally, findings were used to inform recommendations for changes in policy, practice, and education as well as future research specific to improving rural men’s well-being.Health and Social Development, Faculty of (Okanagan)Social Work, School of (Okanagan)Graduat

Prothrombinase assembly on an enveloped virus: evidence that the cytomegalovirus surface contains procoagulant phospholipid

In contrast to quiescent cells, we currently report that purified cytomegalovirus (CMV), strain AD169, constitutively expresses phosphatidylserine (PS)-like procoagulant activity. Initial evidence for this came from the observation of a CMV-dependent decrease in factor Xa clotting times. In a purified system, the assembly of a functional complex between factor Xa and the cofactor Va to form prothrombinase was found to be dependent on the addition of CMV. The corresponding dense bodies (DB) and noninfectious enveloped particles had similar activity. Quantification of the total virion and DB phospholipid, and comparison of prothrombin conversion rates to experiments conducted using known concentrations of PS-containing vesicles showed that 8.5% and 7.2% of the CMV and DB phospholipid, respectively is procoagulant. Direct binding studies of 125I-labeled factor Xa, active site-blocked factor Xa, or the zymogen factor X, with virions or DB showed a single class of Ca(2+)-dependent sites with dissociation constants in the order of 10(-7) MOL/l. Immunogold electron microscopy confirmed the specificity of the CMV/factor Xa association. Cumulatively, these data suggest that the CMV surface contains the necessary procoagulant phospholipid for coagulation enzyme complex assembly. This may enable CMV (and possibly other enveloped viruses) to bypass an important physiologic regulatory mechanism for the production of thrombin.</jats:p