Dynamics of force generation by spreading platelets

Abstract

In order to gain more insight into the role of human platelets for blood clot formation, here we investigate the dynamics of force generation by platelet spreading onto elastic substrates of variable stiffness. Despite their small size, platelets generate high and rapidly varying traction forces on their extracellular environment, which we reconstruct with adapted implementations of Fourier transform traction cytometry. We find that while the final spread area is reached within a few minutes, the build-up of forces typically takes 10-30 minutes. In addition, we identify two distinct behaviors of individual cells, namely oscillating and non-oscillating platelets. An eigenvalue analysis of the platelet dipole tensor reveals a small anisotropy of the exerted force, which is compatible with a random distribution of a few force transmitting centers, in agreement with the observed shapes and traction patterns. We find a correlation between the maximum force level a platelet reaches and its spread area, which we explain by a thin film model for the actively contracting cell. The model reveals a large internal stress of hundreds of kPa. Experimentally we do not find any statistically relevant relation between the force level reached and the substrate stiffness within the stiffness range from 19 to 83 kPa, which might be related to the high platelet activation level used in our study. In addition, our model suggests that due to the uniquely small thickness of platelets, their mechanosensitivity might be limited to a lower stiffness range.

Human blood platelets contract in perpendicular direction to shear flow

“…2,3 When adhering to a substrate, they start to spread to a flat shape, reaching about 30 to 40 mm 2 in area with a height of 100 nm or less. [4][5][6] The activation is initiated via several pathways such as physical triggers, e.g. shear forces, or chemical cues, such as ADP or thrombin.…”

“…The contraction of single blood platelets or small clots in no-flow environments has been thoroughly characterized by different techniques, including micropost arrays, 13,14 atomic force microscopy (AFM), 15 traction force microscopy (TFM) 6,16 and, most recently, tension sensors. 17 The measured forces depend greatly on the method used and can reach some hundreds of nN.…”

“…Further, it was demonstrated that the contractile forces are transmitted at the edge of the platelet. 6,17 To study the influence of shear flow on the contractile behavior of cells, these methods for measuring forces have to be combined with controlled flow fields as provided by microfluidics. Technically, this has been realized in different ways, typically by combining a polydimethylsiloxane (PDMS) structure and a microscopy glass cover slip.…”

Quantifying single‐platelet biomechanics: An outsider’s guide to biophysical methods and recent advances

“…Using polyacrylamide gels with a tunable elastic modulus, Qiu et al observed that fibrinogen functionalized stiffer gels (elastic modulus of 5.0 kPa and 50 kPa) promoted platelet adhesion, spreading, and activation, while on soft gels (0.5 kPa) platelet function was abrogated and was dependent on Rac1 and actomyosin activity. Subsequent TFM experiments on substrates of higher elastic modulus (from 19 kPa up to 83 kPa) revealed that traction forces generated by fully activated platelets were independent of the matrix stiffness . Although platelets generated isotropic contractile traction forces, at the steady state, assessment of force localization showed that these were largest at the periphery of platelets, while the traction force was focused near the platelet granulomere.…”

“…During hemostasis, the ter- revealed that traction forces generated by fully activated platelets were independent of the matrix stiffness. 84 Although platelets generated isotropic contractile traction forces, at the steady state, assessment of force localization showed that these were largest at the periphery of platelets, while the traction force was focused near the platelet granulomere.…”