Binding Site Specificity, Mechanics, and Kinetics of Bimolecular Interactions Underlying Fibrin Polymerization.
Authors: Litvinov Rustem I. , Gorkun Oleg V. , Owen Scott F. , Shuman Henry , Weisel John W. 
Published: October 2019

Abstract
Using laser tweezers-based force spectroscopy, we studied the rupture force profile of complementary interactions between individual fibrin(ogen) molecules and their fragments bearing variable sets of exposed binding sites. The technique of optically trapping and manipulating small particles using a focused laser beam, named laser tweezers, can measure external forces applied to the particle with extremely high resolution and is accurate at the lower end of the force spectrum (0–200 pN), which is suitable to quantify non-covalent intermolecular binding. Accordingly, we have developed an oscillating trap-based model system to study statistical distributions of rupture forces reflecting the whole variety of protein-protein interactions during repeated intermittent contact of surface-bound molecules. Based on several criteria that have been proposed to test whether the observed ruptures were due to the attachments of single or multiple pairs of molecules (PNAS2002, 99:7426; J. Biol. Chem. 2003, 278:51285), our data indicate that under the experimental conditions studied the rupture events are due to single bimolecular attachments. Fibrinogen and fibrinogen fragment D were used as the source of a- and b-holes, and fibrin monomer and fibrin monomer fragment N-DSK (N-terminal disulfide knot) were the source of A- and B-knobs. To distinguish between specific and non-specific binding, two types of control experiments were performed in which the specific interactions were suppressed by either replacing one of the interacting molecules with an inert protein or adding the peptide GPRPam, an inhibitor of fibrin polymerization. Exposure of A-knobs in desA-fibrin or its N-DSK fragment from the central part of the molecule resulted in strong interactions with fibrinogen or fragment D (containing only a- and b-holes), producing a binding strength of ~125–130 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 to 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. Using recombinant fibrinogen mutants with impaired a-sites (γD364H, γD364A) or b-sites (BβD398A) confirmed this conclusion, however, revealed additional weak intermolecular interaction not related to knobs and holes. The A-a interactions had a relatively small zero-force off-rate of ~10−4 s−1 and tight knob-hole contacts characterized by a transition state distance of ~0.3 nm. The results demonstrate that the knob-to-hole binding during thrombin-induced fibrin polymerization is driven by strong, stable, and highly specific A-a bonding, while A-b, B-b, or B-a interactions were not detected. These data provide the molecular mechanisms for mechanical strength and thermodynamic stability of fibrin fibers enabling the accomplishment of physiological functions of a fibrin clot.