Using laser tweezers, we measured for the first time the forces of individual knob-into-hole interactions underlying fibrin polymerization. Exposure of A-knobs in desA-fibrin or its fragment from the central part of the molecule (N-terminal disulphide knot, NDSK) resulted in strong interactions with fibrinogen or fragment D (containing only a- and b-holes), producing a binding strength of approximately 125 to 130 pN. The interactions were not present in the absence of either knobs or holes and were abrogated by a specific inhibitor of fibrin polymerization, a peptide mimic of the A-knob (GPRPam). Exposure of both the A- and B-knobs in desAB-fibrin or desAB-NDSK did not change the rupture force spectra compared with the desA molecules, and their interactions with fibrinogen remained highly sensitive to GPRPam but not to GHRPam (B-knob), suggesting that neither A:b nor B:b nor B:a contacts contributed significantly to binding strength in addition to A:a contacts. The A:a interactions had a relatively small zero-force off-rate of approximately 10(-4) s(-1) and tight knob-to-hole contacts characterized by a transition state distance of approximately 0.3 nm. The results demonstrate that the knob-hole binding during thrombin-induced fibrin polymerization is driven by strong, stable, and highly specific A:a bonding, whereas A:b, B:b, or B:a interactions were not detected.