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

Analysis Method for Measuring Submicroscopic Distances with Blinking Quantum Dots

A method is described that takes advantage of the intermittency (“blinking”) in the fluorescence of quantum dots (QDs) to measure absolute positions of closely spaced QDs. The concept is that even if two QDs are separated by only tens of nanometers, the position of each QD is resolvable if the point spread function of each can be imaged independently of the other. In the case of QDs, this is possible if each QD separately blinks completely on and off during a time-lapse sequence. To demonstrate the principle of this method, time-lapse sequences of single blinking QDs were acquired and the centroids of the point spread functions determined. Images of the blinking QDs were then overlapped in software, pixel by pixel, generating a range of submicroscopic distances between QD pairs. Methods were developed for analyzing the overlapped time sequences of the QD pairs so that the positions of the QDs and the distances between them could be determined without prior knowledge of the single QD positions. We subsequently used this method to measure the end-to-end length of a 122-basepair double-stranded DNA fragment

Kinetics of the Multistep Rupture of Fibrin ‘A-a’ Polymerization Interactions Measured Using Atomic Force Microscopy

Fibrin, the structural scaffold of blood clots, spontaneously polymerizes through the formation of ‘A-a’ knob-hole bonds. When subjected to external force, the dissociation of this bond is accompanied by two to four abrupt changes in molecular dimension observable as rupture events in a force curve. Herein, the configuration, molecular extension, and kinetic parameters of each rupture event are examined. The increases in contour length indicate that the D region of fibrinogen can lengthen by ∼50% of the length of a fibrin monomer before rupture of the ‘A-a’ interaction. The dependence of the dissociation rate on applied force was obtained using probability distributions of rupture forces collected at different pull-off velocities. These distributions were fit using a model in which the effects of the shape of the binding potential are used to quantify the kinetic parameters of forced dissociation. We found that the weak initial rupture (i.e., event 1) was not well approximated by these models. The ruptured bonds comprising the strongest ruptures, events 2 and 3, had kinetic parameters similar to those commonly found for the mechanical unfolding of globular proteins. The bonds ruptured in event 4 were well described by these analyses, but were more loosely bound than the bonds in events 2 and 3. We propose that the first event represents the rupture of an unknown interaction parallel to the ‘A-a’ bond, events 2 and 3 represent unfolding of structures in the D region of fibrinogen, and event 4 is the rupture of the ‘A-a’ knob-hole bond weakened by prior structural unfolding. Comparison of the activation energy obtained via force spectroscopy measurements with the thermodynamic free energy of ‘A-a’ bond dissociation indicates that the ‘A-a’ bond may be more resistant to rupture by applied force than to rupture by thermal dissociation