title: Polymerization of fibrin: Direct observation and quantification of individual B:b knob-hole interactions.
abstract: The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the gamma- and beta-modules. Of the potential knob-hole interactions--A:a, B:b, A:b, and B:a--the first has been shown to be critical for fibrin formation, but the roles 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 A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the gammaD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (beta15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the beta15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.
authors.affiliation: Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1040 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6058, USA. litvinov@mail.med.upenn.edu
authors.forename: Rustem I
authors.initials: RI
authors.lastname: Litvinov
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Gorkun
authors.forename: Dennis K
authors.initials: DK
authors.lastname: Galanakis
authors.forename: Sergiy
authors.initials: S
authors.lastname: Yakovlev
authors.forename: Leonid
authors.initials: L
authors.lastname: Medved
authors.forename: Henry
authors.initials: H
authors.lastname: Shuman
authors.forename: John W
authors.initials: JW
authors.lastname: Weisel
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D005337:Fibrin / Q000737:chemistry*; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry; D005340:Fibrinogen / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D020125:Mutation, Missense; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D052898:Optical Tweezers; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013917:Thrombin / Q000378:metabolism
chemical_list: C115499:BBeta fibrinogen; D005338:Fibrin Fibrinogen Degradation Products; D009842:Oligopeptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C464794:fibrinogen Aalpha; C015214:fibrinogen D fragment; C048770:glycyl-histidyl-arginyl-proline; C034585:glycyl-prolyl-arginyl-proline; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin