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...

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and hematologic disorders such as sickle cell disease (SCD) and malaria often lead to increased endothelial permeability, haemolysis, and microvascular obstruction. However, the associations among these pathological phenomena remain unknown. Here, we report a perfusable, endothelialized microvasculature-on-a-chip featuring an interpenetrating-polymer-network hydrogel that recapitulates the stiffness of blood-vessel intima, basement membrane self-deposition and self-healing endothelial barrier function for longer than 1 month. The microsystem enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found how extracellular heme, a hemolytic byproduct, induces delayed but reversible endothelial permeability in a dose-dependent manner, and demonstrate that endothelial interactions with SCD or malaria-infected erythrocytes cause reversible microchannel occlusion and increased in situ endothelial permeability. The microvasculature-on-a-chip enables mechanistic insight into the endothelial barrier dysfunction associated with SCD, malaria and other inflammatory and haematological diseases.