title: Evidence that fibrinogen γ' directly interferes with protofibril growth: implications for fibrin structure and clot stiffness.
abstract: BACKGROUND
  Fibrinogen contains an alternatively spliced γ-chain (γ'), which mainly exists as a heterodimer with the common γA-chain (γA/γ'). Fibrinogen γ' has been reported to inhibit thrombin and modulate fibrin structure, but the underlying mechanisms are unknown.
  
  
  OBJECTIVE
  We aimed to investigate the molecular mechanism underpinning the influence of γ' on fibrin polymerization, structure and viscoelasticity.
  
  
  METHODS
  γA/γA and γA/γ' fibrinogens were separated using anion exchange chromatography. Cross-linking was controlled with purified FXIIIa and a synthetic inhibitor. Fibrin polymerization was analyzed by turbidity and gel-point time was measured using a coagulometer. We used atomic force microscopy (AFM) to image protofibril formation while final clot structure was assessed by confocal and scanning electron microscopy. Clot viscoelasticity was measured using a magnetic microrheometer.
  
  
  RESULTS
  γA/γ' fibrin formed shorter oligomers by AFM than γA/γA, which in addition gelled earlier. γA/γ' clots displayed a non-homogenous arrangement of thin fibers compared with the uniform arrangements of thick fibers for γA/γA clots. These differences in clot structure were not due to thrombin inhibition as demonstrated in clots made with reptilase. Non-cross-linked γA/γA fibrin was approximately 2.7 × stiffer than γA/γ'. Cross-linking by FXIIIa increased the stiffness of both fibrin variants; however, the difference in stiffness increased to approximately 4.6 × (γA/γA vs. γA/γ').
  
  
  CONCLUSIONS
  Fibrinogen γ' is associated with the formation of mechanically weaker, non-uniform clots composed of thin fibers. This is caused by direct disruption of protofibril formation by γ'.
authors.affiliation: Molecular and Nanoscale Physics Group, School of Physics and Astronomy, University of Leeds, Leeds, UK.
authors.forename: P
authors.initials: P
authors.lastname: Allan
authors.forename: S
authors.initials: S
authors.lastname: Uitte de Willige
authors.forename: R H
authors.initials: RH
authors.lastname: Abou-Saleh
authors.forename: S D
authors.initials: SD
authors.lastname: Connell
authors.forename: R A S
authors.initials: RA
authors.lastname: Ariëns
mesh_terms: D017398:Alternative Splicing; D001777:Blood Coagulation; D001780:Blood Coagulation Tests; D002852:Chromatography, Ion Exchange; D004548:Elasticity; D026122:Factor XIIIa / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism* / Q000648:ultrastructure; D015241:Fibrinogens, Abnormal / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D009391:Nephelometry and Turbidimetry; D058105:Polymerization; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013997:Time Factors; D014783:Viscosity
chemical_list: D015241:Fibrinogens, Abnormal; C527521:fibrinogen gamma'; D005337:Fibrin; D026122:Factor XIIIa