The roles of cofactors protein S and factor V in the TFPI anticoagulant pathway

Tissue factor pathway inhibitor (TFPI) is a principal inhibitor of the initiation of coagulation through inhibition of factor (F) Xa and tissue factor (TF)/FVIIa. While TFPI on its own is a poor inhibitor of FXa, protein S acts as cofactor for TFPI through interaction with TFPI Kunitz 3 domain, greatly enhancing FXa inhibition (~10-fold). The aim of my thesis was to further characterise the molecular basis underlying the TFPI cofactor function of protein S by identifying the complementary functional interaction site on protein S required for TFPI enhancement. I have screened over 40 protein S variants comprising substitutions spanning the whole protein S molecule and have identified the C-terminal sex hormone-binding globulin (SHBG)-like domain to be important for the enhancement of TFPI-mediated inhibition of thrombin generation in plasma. This was demonstrated using a protein S/growth arrest-specific 6 (Gas6) chimera, in which the SHBG-like domain was replaced with the corresponding domain in Gas6. Further examination of this chimera using FXa inhibition assays together with direct binding studies confirmed that the protein S SHBG-like domain is essential for the interaction with TFPI and the enhancement of TFPI-mediated inhibition of FXa. TFPI has also been reported to bind to FV in circulation. In my thesis I have aimed to evaluate the effects of FV interaction with TFPI upon its inhibition of FXa. While FV did not enhance TFPI on its own, it further enhanced the inhibition of FXa by TFPI in the presence of protein S (~32-fold by comparison to TFPI alone), suggesting that FV acts together with protein S as a synergistic cofactor for TFPI. Co-immunoprecipitation experiments revealed a direct interaction between TFPI and FV which was augmented by the presence of protein S. I propose that FV, in cooperation with protein S localise TFPI to the activated membrane surface in a position favourable for the efficient inhibition of FXa. Collectively, my findings shed light on the molecular mechanisms of TFPI enhancement by its cofactors protein S and FV.Open Acces

Protocol of the Cognitive Health in Ageing Register: Investigational, Observational and Trial Studies in Dementia Research (CHARIOT): Prospective Readiness cOhort (PRO) SubStudy

The Cognitive Health in Ageing Register: Investigational, Observational and Trial Studies in Dementia Research (CHARIOT): Prospective Readiness cOhort (PRO) SubStudy (CPSS), sponsored by Janssen Pharmaceutical Research & Development LLC, is an Alzheimer's disease (AD) biomarker enriched observational study that began 3 July 2015 CPSS aims to identify and validate determinants of AD, alongside cognitive, functional and biological changes in older adults with or without detectable evidence of AD pathology at baseline. CPSS is a dual-site longitudinal cohort (3.5 years) assessed quarterly. Cognitively normal participants (60-85 years) were recruited across Greater London and Edinburgh. Participants are classified as high, medium (amnestic or non-amnestic) or low risk for developing mild cognitive impairment-Alzheimer's disease based on their Repeatable Battery for the Assessment of Neuropsychological Status performance at screening. Additional AD-related assessments include: a novel cognitive composite, the Global Preclinical Alzheimer's Cognitive Composite, brain MRI and positron emission tomography and cerebrospinal fluid analysis. Lifestyle, other cognitive and functional data, as well as biosamples (blood, urine, and saliva) are collected. Primarily, study analyses will evaluate longitudinal change in cognitive and functional outcomes. Annual interim analyses for descriptive data occur throughout the course of the study, although inferential statistics are conducted as required. CPSS received ethical approvals from the London-Central Research Ethics Committee (15/LO/0711) and the Administration of Radioactive Substances Advisory Committee (RPC 630/3764/33110) The study is at the forefront of global AD prevention efforts, with frequent and robust sampling of the well-characterised cohort, allowing for detection of incipient pathophysiological, cognitive and functional changes that could inform therapeutic strategies to prevent and/or delay cognitive impairment and dementia. Dissemination of results will target the scientific community, research participants, volunteer community, public, industry, regulatory authorities and policymakers. On study completion, and following a predetermined embargo period, CPSS data are planned to be made accessible for analysis to facilitate further research into the determinants of AD pathology, onset of symptomatology and progression. The CHARIOT:PRO SubStudy is registered with clinicaltrials.gov (NCT02114372). Notices of protocol modifications will be made available through this trial registry. [Abstract copyright: © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

TFPI cofactor function of protein S: essential role of the protein S SHBG-like domain

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI), accelerating the inhibition of activated factor X (FXa). TFPI Kunitz domain 3 residue Glu226 is essential for enhancement of TFPI by protein S. To investigate the complementary functional interaction site on protein S, we screened 44 protein S point, composite or domain swap variants spanning the whole protein S molecule for their TFPI cofactor function using a thrombin generation assay. Of these variants, two protein S/growth arrest-specific 6 chimeras, with either the whole sex hormone-binding globulin (SHBG)-like domain (Val243-Ser635; chimera III) or the SHBG laminin G-type 1 subunit (Ser283-Val459; chimera I), respectively, substituted by the corresponding domain in growth arrest-specific 6, were unable to enhance TFPI. The importance of the protein S SHBG-like domain (and its laminin G-type 1 subunit) for binding and enhancement of TFPI was confirmed in FXa inhibition assays and using surface plasmon resonance. In addition, protein S bound to C4b binding protein showed greatly reduced enhancement of TFPI-mediated inhibition of FXa compared with free protein S. We show that binding of TFPI to the protein S SHBG-like domain enables TFPI to interact optimally with FXa on a phospholipid membrane