Interplay of Platelet Contractility and Elasticity of Fibrin/Erythrocytes in Blood Clot Retraction

Abstract

Blood clot contraction (retraction) is driven by platelet-generated forces propagated by the fibrin network and results in clot shrinkage and deformation of erythrocytes. To elucidate the mechanical nature of this process, we developed a model that combines an active contractile motor element with passive viscoelastic elements. Despite its importance for thrombosis and wound healing, clot contraction is poorly understood. This model predicts how clot contraction occurs due to active contractile platelets interacting with a viscoelastic material, rather than to the poroelastic nature of fibrin, and explains the observed dynamics of clot size, ultrastructure, and measured forces. Mechanically passive erythrocytes and fibrin are present in series and parallel to active contractile cells. This mechanical interplay induces compressive and tensile resistance, resulting in increased contractile force and a reduced extent of contraction in the presence of erythrocytes. This experimentally validated model provides the fundamental mechanical basis for understanding contraction of blood clots.

Figure 1

Model development and comparison with experiments. (a and b) Experimental results (a) and a three-element mathematical model (b) were combined to describe the process of clot contraction. (c) Independent experimental results were used to validate the predictive value of this model. ${\sigma }_{stall}$ and ${\eta }_a$ denote the stall stress and the active viscosity of the platelets (active element), respectively. $K_{fp}$ and $K_{rp}$ denote the bulk modulus of compressed fibrin and RBCs (parallel element), respectively. $K_{fs}$ and $K_{rs}$ denote the bulk modulus of compressed fibrin and RBCs (parallel element), respectively. All of the material properties in the three-element model were fitted using the experimental data, where m represents the matured blood clot and $t_0$ corresponds to the time of clot development. To see this figure in color, go online.

Figure 2

Unconstrained and constrained clot contraction. (a–c) Unconstrained clot contraction was tracked optically (a) to measure changes in clot size over time (b) for paired reconstituted plasma samples without RBCs and with RBCs (c). (d–f) Constrained clot contraction was assessed using a rheometer (d) and after the generation of negative normal stress (e) for paired reconstituted plasma samples without RBCs and with RBCs (f). Data are shown as mean ± SEM at 20 min postactivation. Statistical significance was determined using paired Student’s t-tests with an α-value of 0.05. To see this figure in color, go online.

Figure 3

Active viscoelastic, three-element model for clot contraction. (a and b) The model was developed from scanning electron microscopy images of whole blood clots (a), and each component of the clot was incorporated into the model (b). In (b), platelets (i), fibrin (ii), and RBCs (iii) have been labeled. The three-dimensional model (represented here in one dimension) consists of three elements: an active contractile element made of platelets (ci), and passive parallel (cii) and series (ciii) elements made up of fibrin and RBCs. σ_a is the stress of the active elements, η_a is the viscoelastic coefficient, t₀ is the fibrin network formation time, and K_fp,rp,fs,rs is the bulk modulus, where p represents the parallel element, s represents the series element, r corresponds to RBCs, and f corresponds to fibrin. To see this figure in color, go online.

Figure 4

Clots undergoing unconstrained contraction behave as an active viscoelastic material, not a poroelastic material. (a) Unconstrained clot contraction for volumes ranging from 10 to 100 μL revealed no difference in relative clot size at the measured time points. Statistical significance was assessed using a repeated-measures analysis of variance. (b and c) A computational comparison of the (b) activation time and (c) poroelastic diffusion time revealed that clot contraction is not a result of poroelastic diffusion due to the length scale of the diffusion. The relative volume ratio of the blood clot, $J$, is a function of the scaled radius, $a/R$, and scaled time, $t/t_0$, using the spherically symmetric model for the activation limit (b) and the poroelastic limit (c). Here $R$ and $a$ denote the radial distance from the center and the initial radius of the clot, respectively. To see this figure in color, go online.

Figure 5

Comparison of experimental and modeled clot contraction. (a) Unconstrained clot contraction was tracked for 20 min by recording the change in clot size. Contraction was followed for reconstituted samples with platelets and fibrin alone (labeled without RBCs) and with the addition of RBCs (labeled with RBCs). Likewise, unconstrained contraction was calculated using the three-element model, taking into account the presence and absence of RBCs. (b) Constrained clot contraction was tracked for 20 min by recording the generation of contractile force (negative) using a high-precision rheometer. Contraction was followed for reconstituted samples with platelets and fibrin alone (labeled without RBCs) and with the addition of RBCs (labeled with RBCs). Constrained contraction was calculated using the three-element model, taking into account the presence and absence of RBCs. To see this figure in color, go online.

Figure 6

Validation of the clot contraction model. Unconstrained clot contraction was tracked optically for 20 min at varying volume fractions of RBCs (<10%, 15–20%, 30–40%, and >40%, represented as bars). Data are shown as mean ± SD. The model was used to predict the extent of clot contraction at 20 min for 200,000 μL⁻¹ platelets, 2.5 mg/mL fibrinogen, and varying volume fractions of RBCs. The predicted extent of clot contraction for volume fractions ranging from 0% RBCs to 60% RBCs is represented by the solid line. To see this figure in color, go online.

Figure 7

Effect of RBCs on clot contraction. (a) As time progresses, platelet activation and the development of the fibrin network occur simultaneously. (b–d) The contractile force generated and propagated through the platelet-fibrin meshwork is affected by the presence of RBCs (b), which will ultimately become compacted due to the contractile force, resulting in the volumetric shrinkage of the clot (c). Linear static analysis reveals that larger parallel stiffness, which is due to more blood cells, results in compressive resistance and a reduced degree of clot contraction (d). (e) A larger series stiffness, induced by the presence of more cells, results in a tensile resistance, disruption of the stall stress, and consequently a larger normal force. Too see this figure in color, go online.