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-and-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 D364H 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 mi-metic peptide, the (15-66) 2 fragment containing 2 B-knobs, and a monoclonal anti-body against the 15-21 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. (Blood. 2007;109:130-138)

Mice expressing a mutant form of fibrinogen that cannot support fibrin formation exhibit compromised antimicrobial host defense

“…Our findings are compatible with prior biochemical analyses using snake venom proteases favoring FpA or FpB release, 27,[38][39][40][41] as well as previous reports indicating that polymerization is compromised in the fibrinogen variant g Asp364His , which alters the "a" binding pocket, whereas polymerization is similar to normal in the fibrinogen variant Bb Arg432Ala , which disrupts the "b" binding pocket. [41][42][43][44] B:b interactions are reported to be exceptionally weak as characterized by high affinity constants and a low strength force to rupture the bonds. 43,45 Prior studies suggest any assembly based on cleavage of FpB alone would be restricted to nonphysiologic conditions of low salt concentrations and low temperatures.…”

“…[41][42][43][44] B:b interactions are reported to be exceptionally weak as characterized by high affinity constants and a low strength force to rupture the bonds. 43,45 Prior studies suggest any assembly based on cleavage of FpB alone would be restricted to nonphysiologic conditions of low salt concentrations and low temperatures. 29,39,46 Fib AEK mice and fibrinogen AEK derived from these animals provide novel tools and reagents for more comprehensive studies exploring the consequences of FpA release, FpB release, or both to polymer formation and clot structure both in vitro and in vivo.…”

Effect of Plasmin Treatment on the Fibrin Gel Formation

“…This is the characteristic behavior of fibrin polymerization. In spite that the [18], reptilase-catalyzed fibrin polymerization was carried out as shown in Fig. 2.…”

The Platelet Integrin αIIbβ3 Differentially Interacts with Fibrin Versus Fibrinogen

“…Earlier, this technology enabled us to determine quantitative parameters of integrin-fibrinogen interactions at interfaces and to study their molecular mechanisms (28,29,31,37). Because we can convert fibrinogen immobilized on a surface to fibrin monomer (33,34), we applied this technology to obtain quantitative characteristics and to identify fibrin structures involved in ␣IIb␤3-fibrin interactions. By directly measuring the binding strength between individual fibrin and ␣IIb␤3 molecules, we found clear differences from the interaction of ␣IIb␤3 with fibrinogen.…”

“…To generate beads coated with monomeric fibrin, the fibrinogen-coated beads were treated with human ␣-thrombin (1 unit/ml, 37°C, 1 h) followed by centrifugation and resuspension in 0.1 M HEPES buffer, pH 7.4, containing 150 mM NaCl, 3 mM CaCl 2 , 1 mM MnCl 2 , 2 mg/ml BSA, and 0.1% (v/v) Triton X-100. This treatment has been previously shown to generate biologically active fibrin molecules on a surface (33,34).…”