Quantitative structural mechanobiology of platelet-driven blood clot contraction

Abstract

Blood clot contraction plays an important role in prevention of bleeding and in thrombotic disorders. Here, we unveil and quantify the structural mechanisms of clot contraction at the level of single platelets. A key elementary step of contraction is sequential extension-retraction of platelet filopodia attached to fibrin fibers. In contrast to other cell-matrix systems in which cells migrate along fibers, the "hand-over-hand" longitudinal pulling causes shortening and bending of platelet-attached fibers, resulting in formation of fiber kinks. When attached to multiple fibers, platelets densify the fibrin network by pulling on fibers transversely to their longitudinal axes. Single platelets and aggregates use actomyosin contractile machinery and integrin-mediated adhesion to remodel the extracellular matrix, inducing compaction of fibrin into bundled agglomerates tightly associated with activated platelets. The revealed platelet-driven mechanisms of blood clot contraction demonstrate an important new biological application of cell motility principles.

Fig. 1

Time-lapse images of contracting platelets that cause bending, kinking, and local accumulation of a single fibrin fiber. a Top row: a platelet or a small platelet aggregate (green) attaches to a fiber (red) and spreads filopodia along the fiber axis that contract, inducing a fiber kink and pulling the fiber, compacting it into a dense fibrin knot or coil. For details see Supplementary Movie 1. a Middle row: platelet transformations, including attachment of filopodia to a fiber, spreading and contraction (corresponding to a, top row). a Bottom row: platelet-induced structural changes in a fibrin fiber. The inset (t = 120 s) shows formation of a kink. Arrows indicate filopodia attached to a fiber, including the kink. b Length distributions of the fiber kinks, L_K (n = 42) and platelet filopodia, L_P (n = 300). The bars represent experimental numbers and the curves are log-normal fits. Inset: shortening of a fibrin fiber (black dots) and the shortening rate (blue dotted line) caused by platelet contraction (mean ± SD, n = 30). t*, t** are the microscopic phase transition times separating different regimes of filopodia shortening. c A zoomed fiber kink of a length L_K. d Filopodia with lengths defined as L_P; both parameters presented in b. e Serial images of a contracting platelet reveal reorganization and compaction of fibrin fibers surrounding the cell. Top row: combined fibrin and platelet images; middle row: fibrin network only; bottom row: platelet only. Arrows indicate the platelet’s filopodia attaching to and pulling on fibrin fibers. (see Supplementary Movie 3 for the full sequence at high magnification and Supplementary Movie 4 for an individual platelet filopodium undergoing contraction)

Fig. 2

Platelet-induced fibrin compaction. (a, left): a confocal image of platelets (green) and fibrin (red); (a, center): fibrin fibers and patches (compacted fibrin); (a, right): fibrin co-localized with platelets. Compaction of fibrin by a single cell (S ^SP) and by two platelet aggregates (S ^PA) is shown; scale bar: 3 μm. b, c Changes in the total area of platelet-associated fibrin normalized to its initial value b and the mean absolute area of platelet-co-localized fibrin (c) during the course of clot contraction (mean ± SEM). d Relative portion of fibrin material compacted by platelet aggregates, mean ± SEM, n > 200. The analyzed volume size is 217 µm × 217 µm × 34 µm. e, f Confocal images of a fibrin network in a PRP-clot formed and allowed to contract in the absence (e) and presence (f) of blebbistatin (300 µM). Scale bar: 30 μm

Fig. 3

Clustering of the fibrin-attached neighboring platelets. a Serial confocal images showing formation of secondary platelet clusters due to approximation of platelet-bearing fibrin fibers during clot contraction. Platelets are green and fibrin is red. Scale bar: 5 μm. For the entire sequence, see Supplementary Movies 5, 6. b Platelet area distribution in a contracting clot at different time points with the inset showing the distribution of the larger platelet clusters. c Changes of the average platelet cluster size at different time points of clot contraction corresponding to contraction phases P ₁, P ₂, and P ₃ (mean ± SD, n > 22, *P < 0.05, **P < 0.01, a two-tailed Mann−Whitney test)

Fig. 4

Structural and mechanical contraction kinetics of PRP-clots. a Serial confocal images showing time-dependent densification of the fibrin network; Scale bar: 60 μm. b Dynamic fibrin fluorescence intensity per square micrometer as a measure of fibrin densification (black solid line) characterized by three phases determined by the local extremes of the first derivative (blue dashed line and vertical dashed borders between the phases). The phase designations shown here correspond to the phases P ₁ −P ₄ shown in Fig. 1b. c Fibrin densification as a function of time in the absence and presence of blebbistatin (300 µM) and abciximab (100 µg/ml) (mean ± SD, n = 3). d Fibrin fluorescence intensity (fibrin density) at the end of contraction in the absence and presence of blebbistatin (300 µM) and abciximab (100 µg/ml) (mean ± SD, n = 3, ****P < 0.0001, a two-tailed Mann−Whitney test). e Dynamic contractile stress generated by the platelet-fibrin meshwork in the absence and presence of 100 µg/ml abciximab, a specific antagonist of the platelet receptor integrin αIIbβ3 (averaged kinetic curves, n = 3, mean ± SD). f The kinetic curve shown in e has four phases (P ₁, P ₂ ^A, P ₂ ^B, and P ₃) defined as in b. The first phase, P ₁, corresponds to the pre-contraction stage with the rate constant k ₁; P ₂ ^A and P ₂ ^B comprise the fastest phases with the rate constants k ₂ and k ₃, respectively; and P ₃ is the final contraction phase with the rate constant k ₄. g, h The storage and loss moduli of fully contracted PRP-clots (50 min) in the absence and presence of blebbistatin (300 µM) and abciximab (100 µg/ml) (n = 3, mean ± SD, ****P < 0.0001, a two-tailed Mann−Whitney test)

Fig. 5

Deformation of the platelet-fibrin meshwork during contraction quantified using platelet displacement as a marker indicative of local strain. a, b Representative images of a three-dimensional platelet displacement field of a contracting clot; a top view, fibrin (red), platelet (green), and platelet displacement vectors are shown; b perspective view, platelets (green) with their displacement vectors are visualized. The clot volume analyzed was 217 µm × 217 µm × 34 µm. c, d Changes of the platelet velocity during clot contraction c and clot volume reduction d in the absence and presence of blebbistatin (300 µM) and abciximab (100 µg/ml) (mean ± SD, 310 platelets analyzed in three clots). Statistical significance shown between the volumetric strains of contracted clots in the absence and presence of blebbistatin or abciximab; P < 0.05, Mann−Whitney U-test

Fig. 6

Non-uniform deformation of platelet-fibrin meshwork during clot contraction. Clots were formed on a Triton X-100 pre-coated surface to allow for unconstrained contraction of the clot edges in both vertical and horizontal directions. a Platelet-fibrin mesh z-projections are shown for three different time points during clot contraction and reveal inward movement of the clot edge (white arrow); scale bar: 40 μm. b Three-dimensional (3D) tracking of individual platelets embedded into the fibrin network allow visualization of the 3D platelet displacement field; Scale bar: 40 μm. c A representative contour plot of the mean platelet speed inside a clot (20 µm from the bottom) based on the reconstituted 3D platelet displacement field. The color bar shows the mean speed of platelet displacement (µm/s). d Changes of the platelet moving speed with time during clot contraction, indicating higher speed values at the edge of contracting clots (310 cells in three clots). Data presented in d show the absolute velocity of platelets identified in the temporal sequence of 3D images shown in a. For each time point in d, a set of data points shown corresponds to velocities of platelets tracked in a 3D volume of contracted clot provided in a. As the edge of the clot passes through the view area between 20 and 30 min time points, platelets reach their high velocity values corresponding to a yellow region in the velocity contour plot in c. For the entire sequence of events see Supplementary Movies 7−11