Platelet retraction force measurements using flexible post force sensors

Abstract

Platelets play an important role in hemostasis by forming a thrombotic plug that seals the vessel wall and promotes vascular healing. After platelets adhere and aggregate at the wound site, their next step is to generate contractile forces through the coordination of physicochemical interactions between actin, myosin, and alpha(IIb)beta(3) integrin receptors that retract the thrombus' size and strengthen its adhesion to the exposed matrix. Although platelet contractile forces (PCF) are a definitive feature of hemostasis and thrombosis, there are few approaches that can directly measure them. In this study, we describe the development of an approach to measure PCF in microthrombi using a microscopic flexible post force sensor array. Quasi-static measurements and live microscopic imaging of thrombin-activated platelets on the posts were conducted to assay the development of PCF to various hemostatic conditions. Microthrombi were observed to produce forces that monotonically increased with thrombin concentration and activation time, but forces subsided when thrombin was removed. PCF results were statistically similar on arrays of posts printed with fibronectin or fibrinogen. PCF measurements were combined with clot volume measurements to determine that the average force per platelet was 2.1 +/- 0.1 nN after 60 min, which is significantly higher than what has been measured with previous approaches. Overall, the flexible post arrays for PCF measurements are a promising approach for evaluating platelet functionality, platelet physiology and pathology, the impacts of different matrices or agonists on hemostatic responses, and in providing critical information regarding platelet activity that can guide new hemostatic or thrombotic strategies.

Figure 1

Experimental flow schematic of platelet contractile force measurements. (A) Micropost arrays are printed with matrix ligand and fully submerged in Tyrode buffer. (B) Platelets in Tyrode buffer are added onto the arrays and allowed to settle to the bottom. (C) Thrombin is added to activate the platelets, which induces microthrombus formation and contraction.

Figure 2

Measurement of platelet forces and thrombus volume. (A) Side view image of microthrombus attached to two posts was generated from a Z-stack series of confocal microscopy images. The PDMS of the posts has been stained with DiI (red) and platelet actin with phalloidin (green). The bulk of the thrombus structure lie in between the posts and is connected at the tips of the posts. The volume of the clot is measured by voxel measurements of the actin signal in the Z-stack. (B) Example of contraction force measurements using quasi-static technique. The outline of the clot (yellow line) and its contractile forces (blue arrows) have been determined by image analysis of the post deflections. There is good signal-to-noise in the approach as indicated by the low forces (red arrows) at the posts surrounding the clot.

Figure 3

Platelet contractile forces are activated by thrombin concentration. (A) Total force increases with thrombin concentration on both fibrinogen and fibronectin tip coatings. (B) Clot volume as measured by confocal volume measurements increases with thrombin concentration. (C) Contractile force density is the ratio of total force to clot volume.

Figure 4

Platelet contractile force generation increases steadily with time after thrombin addition. (A) Total force, (B) clot volume, and (C) force density increase in proportion to clotting time.

Figure 5

Washout of freely floating platelets and thrombin causes platelet contractile forces to weaken. (A) Total force, (B) clot volume, and (C) force density decrease with time after washout. Confocal images of representative clots on the arrays are shown for (D) 30 min after activation and (E) 30 additional min after washout (60 min in total after activation).

Figure 6

Live microscopy of thrombus retraction forces. (A) Top row: phase-contrast images of thrombus formation obtained at selected time points. Outline of the clot is shown by yellow trace. Bottom row: fluorescent images of post deflections were used to measure contractile force vectors simultaneously with monitoring of thrombus formation. The clot is not visible under fluorescence, but its outline is shown to illustrate the structural changes in clot formation that accompany the dynamics of contraction force generation. (B) Total force development with time for six clots. Upon thrombin activation at the 25-min mark (arrowhead), all clots exhibit a measureable contractile response. (C) Averaging the total force for all clots in pane B reveals a contractile rate that is similar to the quasi-static measurements shown previously.