Single-molecule force spectroscopy studies of fibrin 'A-a' polymerization interactions via the atomic force microscope

Fibrin, the polymerized form of the soluble plasma protein fibrinogen, plays a critical role in hemostasis as the structural scaffold of blood clots. The primary functions of fibrin are to withstand the shear forces of blood flow and provide mechanical stability to the clot, protecting the wound. While studies have investigated the mechanical properties of fibrin constructs, the response to force of critical polymerization interactions such as the 'A--a' knob--hole interaction remains unclear. Herein, the response of the 'A--a' bond to force was examined at the single-molecule level using the atomic force microscope. Force spectroscopy methodology was developed to examine the 'A--a' interaction while reducing the incidence of both nonspecific and multiple molecule interactions. The rupture of this interaction resulted in a previously unreported characteristic force profile comprised of up to four events. We hypothesized that the first event represented reorientation of the fibrinogen molecule, the second and third represented unfolding of structures in the D region of fibrinogen, and the last event was the rupture of the 'A--a' bond weakened by prior structural unfolding. The configuration, molecular extension, and kinetic parameters of each event in the characteristic pattern were examined to compare the unfolding of fibrin to other proteins unfolded by force. Fitting the pattern with polymer models showed that the D region of fibrinogen could lengthen by ∼50% of the length of a fibrin monomer before rupture of the 'A--a' bond. Analysis showed that the second and third events had kinetic parameters similar to other protein structures unfolded by force. Studies of the dependence of the characteristic pattern on calcium, concentration of sodium chloride, pH, and temperature demonstrated that the incidence of the last event was affected by solution conditions. However, only low pH and high temperatures reduced the probability that an interaction was characteristic, indicating that the force required to rupture the 'A--a' bond was less sensitive than the bond's resilience to structural unfolding to solution conditions. The structural unfolding that precedes the rupture of the 'A--a' bond may prove significant in the polymerization and mechanical properties of fibrin.