Rupture Forces among Human Blood Platelets at different Degrees of Activation

Abstract

Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects.

Figure 1. Characteristics of platelet-surface activation.

SEM toghether with P-selectin and actin indicators show that platelets activate rapidly on PLL (A,E) and Horm collagen (C,G), slower on fibronectin (B,F), while they keep their spherical shape on collagen G (D,H) after 15 min surface-contact. (I) P-selectin level is lowest for platelets on collagen G (black), higher on fibronectin (red), followed by PLL (blue) and highest on Horm collagen (magenta). (J) Spread area of platelets on PLL (blue), fibronectin (red) and Horm collagen (magenta) are significantly higher than on collagen G (black). The thin filopodial extensions and irregular shape are characteristics of activated platelets. Platelets used for calculation: for each experimental condition n = 340 for P-selectin (I) and n = 360 for platelet spread area (J).

Figure 2. Single platelet spreading within 15 min on material-passivated substrates.

(A) Collagen G-passivated tipless cantilever is brought to a loosely bound platelet (red) on collagen G for adhesion. (B) Cantilever with a single platelet is slowly moved up from the substrate. (C) Confocal laser scanning microscopy image of the single platelets stained with Vybrant DiD ex. 644/em.665 (red) at the end of an AFM cantilever. (D) Platelet-probe is brought into contact with material-passivated glass. (E) During activation, platelet spreads and pulls down the cantilever resulting in a spreading force (F_s). (F) The spreading force induced by platelets is low on collagen G (black trace), higher on fibronectin (red trace), and highest on PLL (blue trace).

Figure 3. Schematic illustration of single cell force spectroscopy.

A single platelet (red, a) is brought into contact with another platelet on the substrate (green, A) for interaction (B) as indicated by merging of two colors in the confocal laser scanning microscopy image (C). Cartoon of platelet-platelet interaction, when the platelet on the substrate is adhered to different materials, which results in different degrees of platelet activation and/or release of different molecules (named mediators) (dark cyan in D-F). Mediators become adhesive factors between the platelet on the cantilever and the platelet on the substrate: lowest level on collagen G (D), higher on fibronectin (E), and highest on PLL (F). When the cantilever is separated from the substrate, the adhesion force between two platelets will be measured. (G) Typical retraction curves obtained when the platelet on the substrate is attached to collagen G (black), fibronectin (red), or PLL (blue) showing the short range adhesion forces (F_SR) and long range adhesion forces (F_LR).

Figure 4. Rupture forces among single platelets exposing different degrees of activation.

(A) Histograms showing rupture forces between the platelet-probe and platelet immobilized on collagen G (black), collagen G in the presence of TRAP 6 (grey), fibronectin (red), and PLL (blue). Solid lines are the corresponding Gaussian fits to the data. (B) Average rupture forces with the corresponding standard errors collected from three independent experiments. F_SR = short range forces.

Figure 5. Dissipation works among single platelets exposing different degrees of activation.

(A) Dissipation work required to separate the platelet on the cantilever from the platelets on collagen G (black), collagen G in the presence of TRAP 6 (grey), fibronectin (red), or PLL (blue). (B) Dissipation work obtained from one typical experiment when the basal platelet is immobilized on collagen G (black), collagen G with TRAP 6 (grey), fibronectin (red), or PLL (blue). Solid lines are Gaussian fits.

Figure 6. Rupture forces among single modified platelets.

Rupture force vs rupture distance measured in the absence (black) and presence (red) of abciximab, an inhibitor of GPIIbIIIa. When platelets are preincubated with abciximab, the rupture force reduces on collagen G (A), fibronectin (B), and PLL (C). The corresponding fluorescent images show platelets (cytoskeleton labelled with Phalloidin Atto565) incubated with abciximab on collagen G (D), fibronectin (E), or PLL (F).