An extensional strain sensing mechanosome drives adhesion-independent platelet activation at supraphysiological hemodynamic gradients

Abstract

Background Supraphysiological hemodynamics are a recognized driver of platelet activation and thrombosis at high-grade stenosis and in blood contacting circulatory support devices. However, whether platelets mechano-sense hemodynamic parameters directly in free flow (in the absence of adhesion receptor engagement), the specific hemodynamic parameters at play, the precise timing of activation, and the signaling mechanism(s) involved remain poorly elucidated. Results Using a generalized Newtonian computational model in combination with microfluidic models of flow acceleration and quasi-homogenous extensional strain, we demonstrate that platelets directly mechano-sense acute changes in free-flow extensional strain independent of shear strain, platelet amplification loops, von Willebrand factor, and canonical adhesion receptor engagement. We define an extensional strain sensing “mechanosome” in platelets involving cooperative Ca2+ signaling driven by the mechanosensitive channel Piezo1 (as the primary strain sensor) and the fast ATP gated channel P2X1 (as the secondary signal amplifier). We demonstrate that type II PI3 kinase C2α activity (acting as a “clutch”) couples extensional strain to the mechanosome. Conclusions Our findings suggest that platelets are adapted to rapidly respond to supraphysiological extensional strain dynamics, rather than the peak magnitude of imposed wall shear stress. In the context of overall platelet activation and thrombosis, we posit that “extensional strain sensing” acts as a priming mechanism in response to threshold levels of extensional strain allowing platelets to form downstream adhesive interactions more rapidly under the limiting effects of supraphysiological hemodynamics.

A microfluidic method to investigate platelet mechanotransduction under extensional strain

“…All microfluidic devices were fabricated using established soft photolithography methods [ 18 ]. Briefly, using maskless lithography (MLA 150; Heidelberg instruments), a master template was patterned onto a 4-inch silicon wafer using the SU-8 3050 photoresist (MicroChem Corp) to produce defined channel features with a height of 80 μm.…”

“…The geometry, assumptions employed in the simulation, and boundary conditions can be found in the studies by Zainal Abidin et al. [ 18 ] and Oliveira et al. [ 35 ].…”

“…We recently demonstrated that isolated platelets are capable of directly transducing the extensional strain rate ( ) component of flow acceleration in the absence of canonical adhesion receptor engagement and/or soluble agonist–mediated platelet amplification loops [ 18 ]. We demonstrated that platelets “mechanosense” their environment through membrane delimited extensional strain sensing ( -S) mechanosomes, which comprise Piezo1 as the strain sensor and P2X1 as a Ca 2+ signal amplifier.…”

“…We demonstrated that platelets “mechanosense” their environment through membrane delimited extensional strain sensing ( -S) mechanosomes, which comprise Piezo1 as the strain sensor and P2X1 as a Ca 2+ signal amplifier. We further demonstrated that the class II phosphoinositide 3 kinase C2α isoform acts as a critical regulator of this mechanotransduction mechanism through the modulation of the coupling of extensional force to the mechanosome [ 18 ]. Collectively, these findings raise significant questions regarding the specific nature of fluid-induced forces affecting initial platelet activation and downstream function.…”

“…In addition, common flow-based techniques, such as microcapillary, straight microfluidic, and parallel-plate methods, typically involve mixed kinematics (both shear and extensional components), with most studies only focused on wall shear effects [ 25 ]. To more accurately define the hemodynamic parameters driving the behavior of blood cells and platelets, approaches are shifting from simple, straight channels that provide steady-state laminar shear flow to the use of more complex geometries, such as stepped microcontraction geometries [ 26 ], complex bifurcating microfluidics [ 27 , 28 ], cross-slot devices [ 29 , 30 ], and hyperbolic contractions [ 18 , 31 , 32 ].…”