Mechanics and contraction dynamics of single platelets and implications for clot stiffening

Abstract

Platelets interact with fibrin polymers to form blood clots at sites of vascular injury. Bulk studies have shown clots to be active materials, with platelet contraction driving the retraction and stiffening of clots. However, neither the dynamics of single-platelet contraction nor the strength and elasticity of individual platelets, both of which are important for understanding clot material properties, have been directly measured. Here we use atomic force microscopy to measure the mechanics and dynamics of single platelets. We find that platelets contract nearly instantaneously when activated by contact with fibrinogen and complete contraction within 15 min. Individual platelets can generate an average maximum contractile force of 29 nN and form adhesions stronger than 70 nN. Our measurements show that when exposed to stiffer microenvironments, platelets generated higher stall forces, which indicates that platelets may be able to contract heterogeneous clots more uniformly. The high elasticity of individual platelets, measured to be 10 kPa after contraction, combined with their high contractile forces, indicates that clots may be stiffened through direct reinforcement by platelets as well as by strain stiffening of fibrin under tension due to platelet contraction. These results show how the mechanosensitivity and mechanics of single cells can be used to dynamically alter the material properties of physiologic systems.

Figure 1. Measuring the contraction of single platelets with AFM

a, Cartoon of platelet contraction in a blood clot. The blood clot consists of fibrin gel, single contracting platelets, and platelet aggregates (not shown). Activated platelets adhere to fibrin polymers and contract, driving contraction of the blood clot as a whole. b, Three-dimensional isosurface rendering of multiple confocal microscopy planes shows a single thrombin-activated, membrane fluorescently labelled platelet, attached and spread between a fibrinogen-coated cantilever and a fibrinogen-coated glass surface, simulating the experimental set-up of the side-view AFM system. Scale bar = 1 μm. c, Cartoon of the experimental set-up used to measure single-platelet contraction. A fibrinogen-coated AFM cantilever is pressed slightly against an activated platelet just as it lands on a fibrinogen-coated surface. Contraction of the platelet then pulls the cantilever down towards the surface to a length, L, until the force of the cantilever, F, stalls further contraction. d, Force and platelet length measurement during a typical experiment. Tensional force, or force against platelet contraction due to the cantilever deflection towards the surface, is negative here. e, Zoom in of the first two minutes of the experiment from d showing a small compressional force applied to the platelet to initiate contact, and instantaneous contraction of the platelet.

Figure 2. Stiffness dependence and timescale of platelet contraction

a, Cartoon of isometric clamp experiments that were used to simulate an infinitely stiff environment. As the platelet contracts, the surface is retracted such that the length of the platelet remains constant. b, Typical isometric experiment measurement with the cantilever deflection shown in black and platelet height shown in red. The isometric clamp is turned on after ~2 min. c,d, Distribution of stall forces and contraction rates for platelets pulling against cantilevers with stiffnesses of 18 and 43 pN nm⁻¹, and in an isometric clamp. Medians, quartiles and 90/10 levels are shown, and the * represents a significant difference with a P value of <0.05, ** represents a significant difference with a P value of <0.01, and **** represents a significant difference with a P value of <0.001.

Figure 3. Elasticity and adhesion measurement for contracted platelets

a, Typical elasticity and adhesion measurement. The surface (solid grey line) is ramped out at a speed of 500 nm s⁻¹, or 1,000 nm s⁻¹ for a platelet for which contraction has stalled. The cantilever is pulled down until adhesion to the surface or the cantilever is ruptured, giving the adhesion force. Extensibility is defined as the length of the platelet at the rupture point, relative to its length at the beginning of the ramp-out experiment. b, Stress versus strain during the first 0.5 s of the ramp-out experiment in a. These data are fitted with a line to calculate the elasticity of the contracted platelet. Average measured elasticity was 9.85 kPA (n = 12), average extensibility was 1.57 (n = 11) and adhesion force was 69 nN (n = 11).

Figure 4. Proposed effects of platelets on clot retraction and mechanics

a, During the initial formation of a clot, activated platelets are interspersed in what is probably a heterogeneous fibrin gel. Areas of higher density of fibrin are indicated with darker shading, and exhibit higher stiffnesses. b, The stiffness dependence of platelet contraction probably results in increased forces of contraction in areas of higher stiffness (that is, higher fibrin density), possibly leading to a more uniform contraction of the clot as a whole and an increase in overall elasticity. c, We suggest that the high elasticity of contracted platelets and large adhesion forces between the platelets and the fibrin gel may allow for platelets to reinforce the mechanical properties of the clot directly, by acting as a multi-point crosslinker, restricting deformation and flow of the fibrin gel around the platelet, and by bearing some of the load. Tension on fibrin fibres due to large forces of platelet contraction also may lead to stress stiffening of the fibres under tension, further contributing to stiffening of the clot as a whole.