Fibrin clot structure and mechanics associated with specific oxidation of methionine residues in fibrinogen

Abstract

Using a combination of structural and mechanical characterization, we examine the effect of fibrinogen oxidation on the formation of fibrin clots. We find that treatment with hypochlorous acid preferentially oxidizes specific methionine residues on the α, β, and γ chains of fibrinogen. Oxidation is associated with the formation of a dense network of thin fibers after activation by thrombin. Additionally, both the linear and nonlinear mechanical properties of oxidized fibrin gels are found to be altered with oxidation. Finally, the structural modifications induced by oxidation are associated with delayed fibrin lysis via plasminogen and tissue plasminogen activator. Based on these results, we speculate that methionine oxidation of specific residues may be related to hindered lateral aggregation of protofibrils in fibrin gels.

Figure 1

Fibrinogen molecule with all methionine residues circled and highly oxidized methionine residues labeled. The crystal structure was rendered from the Protein Data Bank (23,24). The α-C domain was added schematically with dotted lines, including the ordered N- and C-terminal subdomains (25).

Figure 2

Absorbance at 350 nm for 1 mg/mL fibrin gels plotted as a function of time during gelation.

Figure 3

SEM images of fibrin gels from HOCl oxidized fibrinogen at 1000× (top) and 20,000× (bottom) magnification.

Figure 4

Elastic (G′) and viscous (G″) moduli of oxidized and baseline fibrin gels plotted as a function of time during gelation.

Figure 5

Linear elastic modulus of the fibrin gels plotted against the fibrinogen concentration.

Figure 6

Instantaneous modulus (G_Inst) of fibrin gels with various concentrations of fibrinogen and oxidized to different levels with 0, 50, and 150 μmol HOCl/g fibrinogen. The data plotted are the average of at least three trials, with error bars corresponding to the standard deviation.

Figure 7

Fibrinolysis induced by tPA and plasminogen in 2.2 mg/mL fibrin gels formed from fibrinogen treated with 0, 50, and 150 μmol/g fibrinogen as monitored by small-amplitude oscillatory rheology. The lysis time for these specific gels is marked with an arrow. The reported lysis times represent the average of three trials.