The interactions between the constituent monomers of fibrin, the polymerized protein network that provides the structural stability of blood clots, are frequently under stress because of the dynamic nature of blood flow. Herein, the calcium dependence of the structural unfolding linked to the forced dissociation of the "A-a" knob-hole bond between fibrin monomers is reported. The presence of calcium was shown to influence the incidence of the last event in the unfolding pattern characteristic of "A-a" rupture. This effect, attributed to the function of the γ1 calcium-binding site, was found to be reversible and specific. Our results indicate that binding of calcium at the γ1 site has no effect on the strength of the knob-hole bond prior to unfolding of the hole-containing γ module. Rather, calcium bound at the γ1 site makes the structure of the hole more resilient to such forced unfolding, leading to survival of the "A-a" knob-hole bond during larger extensions of the fibrinogen molecule but at the cost of rupture of the bond at lower forces.

Fibrin, the polymerized protein that provides the structural scaffold of blood clots, is critical to hemostasis and wound healing. The complexity of the fibrin network has left many questions regarding its formation unanswered, including how temperature, metal concentration, and pH, factors known to mediate of fibrin polymerization, influence the mechanics of the interactions between fibrin monomers. Previously, we used the atomic force microscope to examine the mechanical properties of the 'A–a' knob-hole interaction, the most significant bond between fibrin monomers. Force applied to this bond was shown to cause stepwise unfolding of the hole-bearing region of fibrinogen as evidenced by force curves exhibiting a characteristic pattern of events. The dependence of environmental effects such as pH, temperature, ionic strength, and divalent ion (i.e., Ca2+ and Mg2+) concentration on this characteristic pattern remains unknown. Herein, we examine the force, spacing, and probabilities of each force event in the characteristic pattern as a function of solution chemistry. Calcium concentration significantly influenced incidence of the last event in the characteristic pattern, but otherwise had no effect on the knob-hole interaction. Such behavior, attributed to the high-affinity γ1 calcium-binding site, was found to be reversible and specific. The force data indicate that the γ1 site has no effect on the strength of the knob-hole bond prior to unfolding but makes the hole more resilient to unfolding. Our results may explain previous paradoxical findings that calcium had no affect on the affinity of knob 'A' for hole 'a', but was critical to 'A-a' driven fibrin polymerization. Likewise, understanding the dependence of fibrin mechanics on solution conditions may help resolve other questions surrounding the relationship between the fibrin polymerization and the local solution environment.

Since its invention in 1986 by Binnig, Quate, and Gerber, the atomic force microscope (AFM) has proven to be an extremely useful tool for examining the interactions of proteins with surfaces. Fibrinogen in particular has been used as a model protein to demonstrate new methodologies for studying protein behavior with AFM due to its unique size, shape, and function. Indeed, fibrinogen's central role in both blood coagulation and blood-based infections has made it the primary protein used to interrogate the biocompatibility of surfaces. The goal of this review is to provide an analytical perspective on the utility of AFM for investigating the interaction of fibrinogen with surfaces.

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.

Blood vessel injury triggers the conversion of soluble fibrinogen to insoluble fibrin polymer that serves as the structural scaffold of a blood clot. Understanding the biophysical forces involved in maintaining fibrin structure is of great interest to the biomedical community. Previous reports have identified the 'A-a' interaction as a main contributor to the structural integrity of fibrin. Herein, we present the use of single molecule force spectroscopy to study the forced dissociation of 'A-a' interactions between fibrin molecules. The rupture of the 'A-a' interaction is accompanied by a characteristic force pattern previously unreported in fibrin force spectroscopy, reminiscent of the forced unfolding of other proteins in the literature. We propose that the characteristic pattern represents structural deformation of fibrinogen prior to the rupture of the 'A-a' interaction. Several analysis techniques are employed to characterize each unfolding event of the pattern. First, the polymeric nature of each event is examined using the worm-like chain model. We find that the first three events may be fit with one persistence length, but the significantly larger persistence length of the final event suggests a fundamentally different type of molecular extension than the previous three. Next, the energy landscape of each event is investigated by varying the loading rate. The first event is characterized by an ill-defined force probability distribution, indicating that it might correspond to initial reorientation of the substrate-bound molecule. In contrast, the other events have profiles characteristic of well-defined single-bond ruptures. While high-force events two and three have strikingly similar kinetic parameters implying similar molecular nature, the fourth, low-force event likely represents the final dissociation of the weakened 'A-a' interaction. Characterization of the forced dissociation of 'A-a' interactions may provide insight into the biomechanical properties of fibrin fibers held together via these interactions.

During blood vessel injury, fibrinogen is converted to fibrin, a polymer that serves as the structural scaffold of a blood clot. The primary function of fibrin is to withstand the large shear forces in blood and provide mechanical stability to the clot, protecting the wound. Understanding the biophysical forces involved in maintaining fibrin structure is of great interest to the biomedical community. Previous reports have identified the "A-a" knob-hole interaction as the dominant force responsible for fibrin's structural integrity. Herein, biochemical force spectroscopy is used to study knob-hole interactions between fibrin fragments and variant fibrinogen molecules to identify the forces occurring between individual fibrin molecules. The rupture of the "A-a" knob-hole interaction results in a characteristic profile previously unreported in fibrin force spectroscopy with two distinct populations of specific forces: 110 +/- 34 and 224 +/- 31 pN. In the absence of a functional "A" knob or hole "a", these forces cease to exist. We propose that the characteristic pattern represents the deformation of the D region of fibrinogen prior to the rupture of the "A-a" knob-hole bond.