Submitted July 6, 2006
Accepted August 9, 2006
Polymerization of fibrin: direct observation and quantification of individual B:b knob-hole interactions
Rustem I Litvinov*, Oleg V Gorkun, Dennis K Galanakis, Sergiy Yakovlev, Leonid Medved, Henry Shuman, and John W Weisel
Department of Cell & Developmental Biology, University of Pennsylvania School of Medicine, PA, USA
Department of Pathology and Laboratory Medicine, University of North Carolina, NC, USA
Departments of Pathology and Medicine, School of Medicine, State University of New York, NY, USA
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, MD
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
* Corresponding author; email: litvinov{at}mail.med.upenn.edu.
The polymerization of fibrin occurs primarily via interactions between N-terminal knobs ‘A’ and ‘B’ exposed by cleavage of fibrinopeptides A and B, respectively, and corresponding holes ‘a’ and ‘ b’ in the 
- and 
-modules. Of the potential knob-hole interactions, A:a, B:b, A:b, and B:a, the first one has been shown to be critical for fibrin formation, while the role of the others have remained elusive. Using laser tweezers-based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed knobs ‘ A’ and desB-fibrin bearing knobs ‘ B’ interacted with fragment D from the D364H fibrinogen containing holes ‘b’ but no functional holes ‘a’. The strength of single B:b interactions was found to be 15-20 pN, ~6-fold weaker than A:a interactions. B:b binding was abrogated by the knob ‘B’ mimetic peptide, the (15-66)2 fragment containing two knobs ‘ B’ , and a monoclonal antibody against the 15-21 sequence. The interaction of desB-fibrin with fragment D containing both holes ‘ a’ and ‘ b’ produced the same forces that were insensitive to the knob ‘ A’ mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural knobs ‘ B’ exposed in a fibrin monomer.
