As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin α IIb β 3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation. mechanotransduction | cell mechanics | platelet cytoskeleton | biomaterials A s the first responders at the site of vascular injury, platelets are subjected to a dynamic microenvironment during the process of hemostasis (1-5). Biochemically, platelets are exposed to diverse and rapidly changing gradients of soluble proteins and agonists such as von Willebrand factor, ADP, and thrombin, all of which drive platelet adhesion and activation (6, 7). During this process, platelet activation may take several forms including activation of platelet α IIb β 3 integrins, secretion of α-and dense granules, and membrane phosphatidylserine (PS) exposure leading to a procoagulant phenotype (8, 9). Biophysically, platelets also activate and aggregate in response to the hemodynamic and shear forces of the circulation (10, 11). As clot formation ensues, platelets then interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Although the underlying biochemical signaling pathways that govern the fibrinogen-α IIb β 3 -mediated processes have been well characterized, if and how the mechanical cues in the microenvironment affect platelet activation and physiology remain largely unknown. Indeed, as clot structure and mechanics are known to be heterogeneous within the same clot and more recent studies have demonstrated that platelet activation is also vastly heterogeneous within a growing thrombus (12-14), a systematic approach to investigate how platelet activation is affected by the mechanical microenvironment could lead to profoun...

Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot’s capability to stem haemorrhage are its changing mechanical properties, the major driver of which are the contractile forces exerted by platelets against the fibrin scaffold 1. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding 2 and thrombotic disorders 3. Here, we report a high-throughput hydrogel based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.