Fibrin polymerizes via noncovalent and dynamic association of thrombin-exposed "knobs" with complementary "holes." Synthetic knob peptides have received significant interest as a means for understanding fibrin assembly mechanisms and inhibiting fibrin polymerization. Nevertheless, the inability to crystallize short peptides significantly limits our understanding of knob peptide structural features that regulate dynamic knob:hole interactions. In this study, we used molecular simulations to generate the first predicted structure(s) of synthetic knobs in solution before fibrin hole engagement. Combining surface plasmon resonance (SPR), we explored the role of structural and electrostatic properties of knob "A" mimics in regulating knob:hole binding kinetics. SPR results showed that association rates were most profoundly affected by the presence of both additional prolines as well as charged residues in the sixth to seventh positions. Importantly, analyzing the structural dynamics of the peptides through simulation indicated that the 3Arg side chain orientation and peptide backbone stability each contribute significantly to functional binding. These findings provide insights into early fibrin protofibril assembly dynamics as well as establishing essential design parameters for high-affinity knob mimics that more efficiently compete for hole occupancy, parameters realized here through a novel knob mimic displaying a 10-fold higher association rate than current mimics.
Biomimetics Fibrin