Fibrin network structure and clot mechanical properties are altered by incorporation of erythrocytes

Abstract

Although many in vitro fibrin studies are performed with plasma, in vivo clots and thrombi contain erythrocytes, or red blood cells (RBCs). To determine the effects of RBCs on fibrin clot structure and mechanical properties, we compared plasma clots without RBCs to those prepared with low (2 vol%), intermediate (5-10 vol%), or high (> or =20 vol%) numbers of RBCs. By confocal microscopy, we found that low RBC concentrations had little effect on clot structure. Intermediate RBC concentrations caused heterogeneity in the fiber network with pockets of densely packed fibers alongside regions with few fibers. With high levels of RBCs, fibers arranged more uniformly but loosely around the cells. Scanning electron micrographs demonstrated an uneven distribution of RBCs throughout the clot and a significant increase in fiber diameter upon RBC incorporation. While permeability was not affected by RBC addition, at 20% or higher RBCs, the ratio of viscous modulus (G'') to elastic modulus (G') increased significantly over that of a clot without any RBCs. RBCs triggered variability in the fibrin network structure, individual fiber characteristics, and overall clot viscoelasticity compared to the absence of cells. These results are important for understanding in vivo clots and thrombi.

Figure 1. Confocal images of RBC-containing clots

Clots formed with 0.5 U/ml thrombin, 20 mM CaCl₂, and 0, 2, 10, or 20% RBCs (by volume) with two labels. Alexa 488-labelled fibrin fibers are shown in green, while Vybrant DiD-labelled RBC membranes are shown in red. A) 20X, magnification bar = 50 μm; B) 126X, magnification bar = 10 μm.

Figure 2. Scanning electron micrographs of RBC-containing clots

Clots formed with 0%, 2%, 10%, and 20% RBCs (by volume) with 20 mM CaCl₂ and 0.5 U/ml thrombin. Clots were prepared in chambers that were rotated during clot formation, washing, and fixing to prevent RBC settling. Images shown in this figure were taken at 1,000X; magnification bar = 20 μm.

Figure 3. Permeability of RBC-containing clots

Clot permeability was measured by flow of HBS through a clot for 30 min. Free RBCs were washed out with HBS prior to measurement. The permeability constant was calculated according to the Darcy equation (see Methods). Data are plotted as mean ± standard error of the mean (n=5–8), and p > 0.15 for all comparisons (independent t-test).

Figure 4. Viscoelastic measurements of RBC-containing clots

Viscoelastic measurements were performed on clots formed from PPP with 0 – 50% RBCs (by volume) with 20 mM CaCl₂ and 1 U/ml thrombin on a TA Instruments RFS II Rheometer. G′ and G″ values are shown in plot A, and tan δ values are shown in plot B. Data are plotted as mean ± standard error of the mean (n=5–10). * p < 0.05 for comparison with 0% RBCs (using independent t-test).