Kinetics of the multistep rupture of fibrin 'A-a' polymerization interactions measured using atomic force microscopy

Abstract

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 approximately 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.

Figure 1

(A) Schematic of AFM experimental configuration. Space-filling models of fibrin fragment desAB-NDSK and fibrinogen colored by polypeptide chains α, β, and γ, and available in an online version of the article. The formation of an ‘A-a’ bond (circled) is shown between the γ-module of fibrinogen and knob ‘A’ of the desAB-NDSK fragment (dashed line). Knob ‘A’ (dashed line) does not appear in crystal structures and is thus approximated. Fibrin(ogen) knob ‘B’ and αC domains are not shown. (B) Detailed representation of ‘A-a’ bond showing fibrinogen D region (ribbons) with bound GPRP peptide (spheres). The GPRP peptide is knob ‘A’ peptide-mimetic. The following Protein Data Bank entries were used to generate protein models: 3GHG (fibrinogen), 2A45 (desAB-NDSK), 1BJ5 (BSA), and 1LTJ (fibrinogen D region with bound knob ‘A’). Scheme is not to scale. Protein models were generated with Pymol (DeLano Scientific, Palo Alto, CA).

Figure 2

Force curves (restoring force in pN versus tip-substrate separation in nm) containing characteristic pattern of fibrin ‘A-a’ knob-hole forced dissociation. Four types of characteristic patterns were identified: doublet (A), doublet with preceding event (B), doublet with following event (C), and doublet with both preceding and following events (D). Event numbers are indicated. Linear approximation of slope before one event, as used for loading rate calculation, is shown as dashed gray line (the line is slightly offset for clarity).

Figure 3

(Left) Schematic of parallel, series, and zipper bond configurations. The force experienced by each interaction (F₁ represents the first bond to rupture and F₂, the second), relative to total applied force (F), is indicated. (Right) Hypothetical force curves for each configuration, showing the total applied force (solid), F₁ (dotted), and F₂ (dashed). Select changes in restoring force in the force curves that correlate with the changes in force applied to each bond are indicated.

Figure 4

Probability distributions of the rupture forces in a representative experiment with tip retraction velocity of 1 μm/s and cantilever stiffness of 55 pN/nm.

Figure 5

Representative four-event characteristic pattern force curve (circles) fit with the freely jointed chain model (lines). Least-square fits with event 1 in parallel (gray) and in series (black) with event 2 are shown. Events 2–4 were always fit in series configuration.

Figure 6

Characteristic force patterns of the rupture of interaction between fibrinogen and desAB-NDSK, where the rupture force is plotted versus relative separation (A) and versus relative contour length extracted from FJC fitted curves with events 1 and 2 modeled in parallel (B) and in series or zipper configuration (C).

Figure 7

(A) Dissociation rates as a function of force for each event as it occurs in curves with just events 2 and 3 (▵), events 1–3 (▿), events 2–4 (^∗), and all four events (○), as identified in Fig. 2, where error bars represent error of the mean among experiments. (B) Dissociation rates as a function of force for each event, with curve types averaged and weighted by error (circles) and fit with Eq. 3 (line).