1. Cell Biology
  2. Structural Biology and Molecular Biophysics

Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability and resistance to fibrinolysis

  1. Helen McPherson
  2. Cedric Duval
  3. Stephen R Baker
  4. Matthew S Hindle
  5. Lih T Cheah
  6. Nathan L Asquith
  7. Marco M Domingues
  8. Victoria C Ridger
  9. Simon DA Connell
  10. Khalid Naseem
  11. Helen Philippou
  12. Ramzi A Ajjan
  13. Robert AS Ariens  Is a corresponding author
  1. University of Leeds, United Kingdom
  2. Wake Forest University, United States
  3. Harvard Medical School, United States
  4. Universidade de Lisboa, Portugal
  5. University of Sheffield, United Kingdom
  6. University of Leeds, United States
https://doi.org/10.7554/eLife.68761
Share this article
Cite this article
  1. Helen McPherson
  2. Cedric Duval
  3. Stephen R Baker
  4. Matthew S Hindle
  5. Lih T Cheah
  6. Nathan L Asquith
  7. Marco M Domingues
  8. Victoria C Ridger
  9. Simon DA Connell
  10. Khalid Naseem
  11. Helen Philippou
  12. Ramzi A Ajjan
  13. Robert AS Ariens
(2021)
Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability and resistance to fibrinolysis
eLife 10:e68761.
https://doi.org/10.7554/eLife.68761
  2. Download BibTeX
  3. Download .RIS

Abstract

Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis.

Data availability

The source data for Figures 1 B-F, figure 2 B and D, figure 3 B, figure 4, figure 5 B, C and D and figure 6 A-C and D-F and supplementary Figures 1 supplement 1, figures 4 supplement 1 and figures 5 supplement 1 and 2 and figures 6 supplement 1 are made available as separate source data files.

Article and author information

Author details

  1. Helen McPherson

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3519-498X

  2. Cedric Duval

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  3. Stephen R Baker

    Department of Physics, Wake Forest University, Winston Salem, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3147-4925

  4. Matthew S Hindle

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  5. Lih T Cheah

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  6. Nathan L Asquith

    Division of Hematology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.

  7. Marco M Domingues

    Instituto de Medicina Molecular - João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisbon, Portugal
    Competing interests
    The authors declare that no competing interests exist.

  8. Victoria C Ridger

    Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  9. Simon DA Connell

    Molecular and Nanoscale Physics Group, University of Leeds, Leeds, United States
    Competing interests
    The authors declare that no competing interests exist.

  10. Khalid Naseem

    Discovery and Translational Science Department, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  11. Helen Philippou

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  12. Ramzi A Ajjan

    Discovery and Translational Science Department, Leeds Institute of Cariovasular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1636-3725

  13. Robert AS Ariens

    Discovery anTranslational Science Department, University of Leeds, Leeds, United Kingdom
    For correspondence
    R.A.S.Ariens@leeds.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6310-5745

Funding

British Heart Foundation (RG/13/3/30104)

  • Helen McPherson
  • Cedric Duval
  • Stephen R Baker
  • Marco M Domingues
  • Victoria C Ridger
  • Simon DA Connell
  • Helen Philippou
  • Ramzi A Ajjan
  • Robert Ariens

British Heart Foundation (RG/18/11/34036)

  • Helen McPherson
  • Cedric Duval
  • Stephen R Baker
  • Victoria C Ridger
  • Simon DA Connell
  • Helen Philippou
  • Ramzi A Ajjan
  • Robert Ariens

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: Procedures were performed according to accepted standards of humane animal care, approved by the ethical review committee at the University of Leeds, and conducted under license (P144DD0D6) from the United Kingdom Home Office.

Copyright

© 2021, McPherson et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,900
    views
  • 255
    downloads
  • 22
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Helen McPherson
  2. Cedric Duval
  3. Stephen R Baker
  4. Matthew S Hindle
  5. Lih T Cheah
  6. Nathan L Asquith
  7. Marco M Domingues
  8. Victoria C Ridger
  9. Simon DA Connell
  10. Khalid Naseem
  11. Helen Philippou
  12. Ramzi A Ajjan
  13. Robert AS Ariens
(2021)
Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability and resistance to fibrinolysis
eLife 10:e68761.
https://doi.org/10.7554/eLife.68761

Share this article

https://doi.org/10.7554/eLife.68761

Categories and tags

Research organisms

Further reading

    1. Cell Biology
    2. Immunology and Inflammation

    UFMTrack, an Under-Flow Migration Tracker enabling analysis of the entire multi-step immune cell extravasation cascade across the blood-brain barrier in microfluidic devices

    Mykhailo Vladymyrov, Luca Marchetti ... Britta Engelhardt
    Tools and Resources

    The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multistep T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro. Here, we present the under-flow migration tracker (UFMTrack) framework for studying immune cell interactions with endothelial monolayers under physiological flow. We then showcase a pipeline built based on it to study the entire multistep extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro. UFMTrack achieves 90% track reconstruction efficiency and allows for scaling due to the reduction of the analysis cost and by eliminating experimenter bias. This allowed for an in-depth analysis of all behavioral regimes involved in the multistep immune cell extravasation cascade. The study summarizes how UFMTrack can be employed to delineate the interactions of CD4+ and CD8+ T cells with the BBB under physiological flow. We also demonstrate its applicability to the other BBB models, showcasing broader applicability of the developed framework to a range of immune cell-endothelial monolayer interaction studies. The UFMTrack framework along with the generated datasets is publicly available in the corresponding repositories.

    1. Cell Biology
    2. Chromosomes and Gene Expression

    Treacle’s ability to form liquid-like phase condensates is essential for nucleolar fibrillar center assembly, efficient rRNA transcription and processing, and rRNA gene repair

    Artem K Velichko, Nadezhda V Petrova ... Omar L Kantidze
    Research Article

    We investigated the role of the nucleolar protein Treacle in organizing and regulating the nucleolus in human cells. Our results support Treacle’s ability to form liquid-like phase condensates through electrostatic interactions among molecules. The formation of these biomolecular condensates is crucial for segregating nucleolar fibrillar centers from the dense fibrillar component and ensuring high levels of ribosomal RNA (rRNA) gene transcription and accurate rRNA processing. Both the central and C-terminal domains of Treacle are required to form liquid-like condensates. The initiation of phase separation is attributed to the C-terminal domain. The central domain is characterized by repeated stretches of alternatively charged amino acid residues and is vital for condensate stability. Overexpression of mutant forms of Treacle that cannot form liquid-like phase condensates compromises the assembly of fibrillar centers, suppressing rRNA gene transcription and disrupting rRNA processing. These mutant forms also fail to recruit DNA topoisomerase II binding protein 1 (TOPBP1), suppressing the DNA damage response in the nucleolus.

    1. Cell Biology

    Myristoylated Neuronal Calcium Sensor-1 captures the preciliary vesicle at distal appendages

    Tomoharu Kanie, Roy Ng ... Peter K Jackson
    Research Article Updated

    The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of preciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures preciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the preciliary vesicle recruitment, but not for other steps of cilium formation (Kanie et al., 2025). The lack of a membrane-binding motif in CEP89 suggests that it may indirectly recruit preciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and the centriole-associated vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similar to CEP89 knockouts, preciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the preciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the preciliary vesicles.