Fibrinogen variant BbetaD432A has normal polymerization but does not bind knob "B".
Fibrinogen residue Bbeta432Asp is part of hole "b" that interacts with knob "B," whose sequence starts with Gly-His-Arg-Pro-amide (GHRP). Because previous studies showed BbetaD432A has normal polymerization, we hypothesized that Bbeta432Asp 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 BbetaD432A. Surprisingly, the structure (rfD-BbetaD432A+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 BbetaD432A were indistinguishable compared with normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BbetaD432A 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.
Bowley
Sheryl R
SR
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-7525, USA.
Lord
Susan T
ST
D000818:Animals; D012103:Batroxobin / Q000494:pharmacology; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D005176:Factor XIII / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006490:Hemostatics / Q000494:pharmacology; D006801:Humans; D008855:Microscopy, Electron, Scanning; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
C115499:BBeta fibrinogen; D003432:Cross-Linking Reagents; D005343:Fibrinolytic Agents; D006490:Hemostatics; D009842:Oligopeptides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C048770:glycyl-histidyl-arginyl-proline; D005337:Fibrin; D005340:Fibrinogen; D005176:Factor XIII; D012103:Batroxobin; D013917:Thrombin; D002118:Calcium