High-Resolution Integrin Molecular Tension Dynamics during Platelet Adhesion and Activation

Abstract

Platelets are small disc-shaped cell fragments circulating in the bloodstream. Upon injury, platelets adhere and aggregate on injured subendothelium to form blood clots to stop bleeding. Irregular platelet adhesion and aggregation may cause acute coronary syndrome or other cardiovascular diseases. Membrane protein integrins mediate platelet adhesion and transmit tensions to activate platelets, therefore playing important roles in platelet functions. Nevertheless, cellular force study in platelets is very limited and integrin molecular tensions were never calibrated in platelets, presumably due to the 2∼3 µm size of platelets which is at the resolution limit of conventional cell traction force microscopy. Here we applied a novel molecular tension sensor and modulator named TGT (tension gauge tether) to study integrin tensions in platelets. TGT is a rupturable molecular linker with a programmable tension tolerance (Ttol). As a tension modulator, TGT globally restricts integrin tensions under the designed level of Ttol, enabling the study of integrin-tension dependency for a certain cellular function. As a tension sensor, TGT maps integrin tensions by fluorescence with a spatial resolution of 0.2 µm, ten times higher than conventional methods. Using TGT, we determined that platelet adhesion and spreading requires integrin tension lower than 12 pN, a force much less than the ∼40 pN integrin tension required by regular mammalian cell adhesion. This surprising result suggests that platelets have extraordinary adhesion and spreading ability which might be beneficial for the rapid response of platelets to injuries. We also monitored calcium oscillation in platelets to confirm that platelets are activated at this low integrin tension level. Next, using TGT as a tension sensor, we provided the high-resolution (0.2 µm) integrin tension map for platelet adhesion for the first time. Real-time tension mapping shows that integrin tensions at a high level (>54 pN) are initially concentrated in one or two micron-sized punctate regions upon platelet adhesion. After full platelet spreading, these integrin tensions spread out to the entire platelet-substrate interface and the tension level decreases into the range of 12∼54 pN. These high integrin tensions are not required for platelet adhesion and activation, but may be required for platelet contraction, as these tensions were abolished by myosin II inhibition without compromising platelet adhesion and activation. Overall, our research has initiated the biomechanical study of platelets at the molecular tension level, and revealed a rich dynamics of integrin tensions in platelets.