Mechanical stability and fibrinolytic resistance of clots containing fibrin, DNA, and histones

Abstract

Neutrophil extracellular traps are networks of DNA and associated proteins produced by nucleosome release from activated neutrophils in response to infection stimuli and have recently been identified as key mediators between innate immunity, inflammation, and hemostasis. The interaction of DNA and histones with a number of hemostatic factors has been shown to promote clotting and is associated with increased thrombosis, but little is known about the effects of DNA and histones on the regulation of fibrin stability and fibrinolysis. Here we demonstrate that the addition of histone-DNA complexes to fibrin results in thicker fibers (increase in median diameter from 84 to 123 nm according to scanning electron microscopy data) accompanied by improved stability and rigidity (the critical shear stress causing loss of fibrin viscosity increases from 150 to 376 Pa whereas the storage modulus of the gel increases from 62 to 82 pascals according to oscillation rheometric data). The effects of DNA and histones alone are subtle and suggest that histones affect clot structure whereas DNA changes the way clots are lysed. The combination of histones + DNA significantly prolongs clot lysis. Isothermal titration and confocal microscopy studies suggest that histones and DNA bind large fibrin degradation products with 191 and 136 nM dissociation constants, respectively, interactions that inhibit clot lysis. Heparin, which is known to interfere with the formation of neutrophil extracellular traps, appears to prolong lysis time at a concentration favoring ternary histone-DNA-heparin complex formation, and DNase effectively promotes clot lysis in combination with tissue plasminogen activator.

Figures

FIGURE 1.

Fibrin, histone, and DNA content of arterial thrombi. Following thrombectomy, thrombus samples were either frozen for immunostaining or washed, fixed, and dehydrated for SEM processing as detailed under “Experimental Procedures.” Sections of frozen samples were doubly immunostained for fibrin (green) and histone 1 (red) as well as with a DNA dye, TOTO-3 (blue). Images were taken at an original magnification of ×20 with a confocal laser microscope. SEM images were taken from the fixed samples of the same thrombi. TO, a thrombus from popliteal artery; GI, a thrombus from infrarenal aorta aneurysm; TJ, a thrombus from femoropopliteal graft. Scale bars, 2 μm in SEM panels and 50 μm in all other panels.

FIGURE 2.

Small angle x-ray scattering of fibrin clots containing 100 μg/ml DNA, 300 μg/ml histone, 10 IU/ml heparin, or their combinations at the same concentrations. Curves are shifted vertically by the factors indicated at their origin for better visualization. Symbols represent the measured intensity values, and solid lines show the fitted empirical functions as described in the supplemental table. The dashed vertical line indicates the longitudinal periodicity of about 22 nm, and the solid vertical lines show the boundaries of the broad peaks that characterize the lateral structure of the fibrin fibers.

FIGURE 3.

Rheology studies showing the effect of DNA, histones, and DNA-histone on the critical shear stress needed to disassemble fibrin. Curves are shown for pure fibrin (red), fibrin containing 50 μg/ml DNA (green), and 100 μg/ml DNA (magenta). The addition of histones leads to increased clot stability under sheer stress shown in the presence of 300 μg/ml histone (blue) and 300 μg/ml histone + 100 μg/ml DNA (black). Pa, pascals.

FIGURE 4.

Effect of DNA and histones included within fibrin clots on clotting and lysis profiles.A shows the effects of 0 (circles), 0.03 (squares), 0.13 (triangles), and 0.5 mg/ml (diamonds) DNA on clotting and lysis in the absence of histones. B shows the effects of 0 (circles), 0.04 (squares), 0.33 (triangles), and 1 mg/ml histones (diamonds) in the absence of DNA. C shows the same range of histones as in B but in the presence of 0.03 mg/ml DNA. In all cases, only every tenth point is shown for clarity

FIGURE 5.

Effects of DNA and histones on appearance of fibrin clots and rates of lysis. Clot lysis was monitored in the presence of (final concentrations) 1.4 mg/ml fibrinogen and 200 nm plasminogen clotted with 30 nm thrombin in the presence of 0.2 nm tPA. Tubes in positions 1–5 have the following additions: none, 50 μg/ml histones, 70 μg/ml DNA, DNA + histones, DNA + histones + 5 units/ml DNase I, respectively. In A, the DNase was separated from the DNA until clotting was initiated, and in B, the DNA solution was pretreated with DNase I for 30 min before clotting. The reaction was monitored for 3 h during which 2000 pictures were taken to form a time lapse video (supplemental Videos 1 and 2). The images shown here were selected at the times given to illustrate significant stages of the reaction.

FIGURE 6.

Confocal microscopy studies using tPA-GFP and red fluorescent fibrin after 25 min of fibrinolysis. Each column of micrographs from left to right shows green tPA-GFP fluorescence, red Alexa Fluor 546-conjugated fibrin fluorescence, and the merged image. The first row shows the accumulation of fibrin aggregates that co-localize with tPA-GFP. The second row with the addition of 70 μg/ml DNA shows less fibrin aggregate formation but a diffuse fibrin clot that remains behind the advancing tPA-GFP front. The lower two rows where clots contain 50 μg/ml histones and 50 μg/ml histones + 70 μg/ml DNA, respectively, demonstrate reduced formation of fibrin aggregates within fibrin and less binding of tPA-GFP. The numbers in the last column indicate the relative distance for penetration of tPA-GFP in the clot at 25 min (the mean value for pure fibrin is 1, mean and S.E. from at least six samples, p < 0.05 for all additives according to the Kolmogorov-Smirnov test in comparison with pure fibrin). Scale bars, 20 μm.

FIGURE 7.

Binding of FDPs and DNA studied using ITC. The cell (1.43 ml) of the titration calorimeter was filled with 0.5 mg/ml DNA, 25 successive aliquots (10 μl each) of 6 μm FDPs were injected into the cell at 25 °C, and the heat increments of each addition (raw differential power (DP)) were measured (top panel). The base line-corrected, peak-integrated, and concentration-normalized enthalpy changes (ΔQN; bottom panel, squares) were evaluated according to the single site algorithm, and the best fitting binding isotherm is shown. The inset shows a non-reducing SDS-PAGE gel of typical FDP preparations consisting of high molecular weight fibrin fragments (binding) and low molecular weight fibrin fragments (non-binding).

FIGURE 8.

Heparin displaces DNA in the histone-DNA complex.A shows the effect of unfractionated heparin concentration on maximum clot absorbance for clots containing 0.015 mg/ml DNA and 0 (crosses), 0.02 (circles), 0.05 (diamonds), 0.1 (triangles), 0.25 (squares), and 0.3 mg/ml (inverted triangles) histone. B–F show the effects of increasing unfractionated heparin concentration on the times to 50% lysis (not with same ranges but all in seconds) in clots containing 0.015 mg/ml DNA and the same range of histone concentrations as in A. In all cases, the crosses show the effect of heparin alone (lysis times with DNA but no histones), and the x axis is heparin concentration (log scale IU/ml in reaction mixture).