Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.
BACKGROUND
The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin.


OBJECTIVE
We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together.


METHODS
We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK.


RESULTS
The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK.


CONCLUSIONS
The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.
Geer
C B
CB
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.
Tripathy
A
A
Schoenfisch
M H
MH
Lord
S T
ST
Gorkun
O V
OV
D000327:Adsorption; D012103:Batroxobin / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D005340:Fibrinogen / Q000737:chemistry / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008954:Models, Biological; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000378:metabolism*
D009842:Oligopeptides; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C048770:glycyl-histidyl-arginyl-proline; C034585:glycyl-prolyl-arginyl-proline; D005340:Fibrinogen; D012103:Batroxobin; D013917:Thrombin