Fibrinogen variant BβD432A has normal polymerization but does not bind knob "B"

Fibrinogen residue B beta 432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-ArgPro-amide (GHRP). Because previous studies showed B beta D432A has normal polymerization, we hypothesized that B beta 432Asp is not critical for knob "B" binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from B beta D432A. Surprisingly, the structure (rfD-B beta D432A+GH) showed the peptide GHRP was not bound to hole "b." We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure, and the rate of FXIIIa cross-linking. The turbidity and the rate of gamma-gamma dimer formation for B beta D432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of B beta D432A and normal, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than "B:b" interactions. We conclude that hole "b" and "B:b" knob-hole binding per se have no influence on fibrin polymerization. (Blood. 2009; 113: 4425-4430)