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

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

  • 1,819
    views
  • 244
    downloads
  • 19
    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

Further reading

    1. Cell Biology
    Tomoharu Kanie, Beibei Liu ... Peter K Jackson
    Research Article

    Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.

    1. Cell Biology
    Tomoharu Kanie, Roy Ng ... Peter K Jackson
    Research Article

    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 ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary 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 a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary 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 ciliary vesicles.