Complexity of “A−a” Knob−Hole Fibrin Interaction Revealed by Atomic Force Spectroscopy

Abstract

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.

The molecular origins of the mechanical properties of fibrin

“…Recent studies by Averett et al provide direct evidence that the γ-nodules unfold in response to mechanical stress (33, 34). These authors developed a novel method to measure the forced dissociation of ‘A:a’ interaction using AFM.…”

“…In subsequent studies of multi-step ‘A:a’ bond rupture, Averett et al demonstrated that the force applied to hole ‘a’ through bound knob ‘A’ induced stepwise unfolding of the γ-nodule prior to rupture of the ‘A:a’ bond (33, 34). Remarkably, the strength of the ‘A:a’ bond was sufficient to survive through the unfolding events.…”

“…This model follows from earlier work that showed forced unfolding of the coiled-coils in single molecules or small oligomers of fibrinogen (11, 12). Two recent papers by Gorkun and colleagues have shown the ‘A:a’ interactions that mediate protofibril formation are sufficiently strong to unfold the γ-nodule prior to breaking this knob-hole bond (33, 34). These experiments suggest unfolding of the γ-nodules occurs under strain.…”

Distributions of Parameters and Features of Multiple Bond Ruptures in Force Spectroscopy by Atomic Force Microscopy

“…Also, a characteristic rupture pattern is observed in rupture of the molecular bond responsible for initial polymerization of fibrin. 21 For these interactions, it is suggested that the characteristic pattern consisting of two, four sequential ruptures originates from unfolding of a globular D domain of fibrin. A single-molecule nature of these ruptures is supported by the analysis of distributions of distances between rupture events and by the detailed kinetic analysis of distributions of rupture forces.…”

“…This fingerprint is often obtained from mechanical unfolding of "polyproteins", [17][18][19][20] or it naturally occurs during rupture of molecular bonds. 21,22 Stretching of a polyprotein with individual "monomers" corresponding to folded globular proteins shows the restoring force that exhibits a sawtooth-like rupture pattern. In this pattern, the peak force is interpreted as unfolding of individual domains, and the spacing between the peaks corresponds to the gain in length of unfolded regions.…”