Yongzhi Qiu

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

Significance Platelets are cell fragments in the blood that initiate clot formation at the site of bleeding. Although the biological aspects of this process have been well characterized, whether platelets can detect and physiologically respond to the mechanical aspects of its local environment is unclear. Here, we show that platelets sense the stiffness of the underlying clot substrate, and increasing substrate stiffness increases platelet adhesion and spreading. Importantly, adhesion on stiffer substrates leads to higher levels of platelet activation. Mechanistically, we determined that Rac1, actin, and myosin activity mediate this process. This newfound capability of how platelets adjust their degree of activation based on the mechanical properties of their environment provides new insight into how clots are formed. 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 αIIbβ3 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.

Single-platelet nanomechanics measured by high-throughput cytometry

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.

Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions

The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.

Platelets and physics: How platelets “feel” and respond to their mechanical microenvironment

During clot formation, platelets are subjected to various different signals and cues as they dynamically interact with extracellular matrix proteins such as von Willebrand factor (vWF), fibrin(ogen) and collagen. While the downstream signaling of platelet-ligand interactions is well-characterized, biophysical cues, such as hydrodynamic forces and mechanical stiffness of the underlying substrate, also mediate these interactions and affect the binding kinetics of platelets to these proteins. Recent studies have observed that, similar to nucleated cells, platelets mechanosense their microenvironment and exhibit dynamic physiologic responses to biophysical cues. This review discusses how platelet mechanosensing is affected by the hydrodynamic forces that dictate vWF-platelet interactions and fibrin polymerization and network formation. The similarities and differences in mechanosensing between platelets and nucleated cells and integrin-mediated platelet mechanosensing on both fibrin(ogen) and collagen are then reviewed. Further studies investigating how platelets interact with the mechanical microenvironment will improve our overall understanding of the hemostatic process.

Biomechanics of haemostasis and thrombosis in health and disease: from the macro- to molecular scale.

Although the processes of haemostasis and thrombosis have been studied extensively in the past several decades, much of the effort has been spent characterizing the biological and biochemical aspects of clotting. More recently, researchers have discovered that the function and physiology of blood cells and plasma proteins relevant in haematologic processes are mechanically, as well as biologically, regulated. This is not entirely surprising considering the extremely dynamic fluidic environment that these blood components exist in. Other cells in the body such as fibroblasts and endothelial cells have been found to biologically respond to their physical and mechanical environments, affecting aspects of cellular physiology as diverse as cytoskeletal architecture to gene expression to alterations of vital signalling pathways. In the circulation, blood cells and plasma proteins are constantly exposed to forces while they, in turn, also exert forces to regulate clot formation. These mechanical factors lead to biochemical and biomechanical changes on the macro‐ to molecular scale. Likewise, biochemical and biomechanical alterations in the microenvironment can ultimately impact the mechanical regulation of clot formation. The ways in which these factors all balance each other can be the difference between haemostasis and thrombosis. Here, we review how the biomechanics of blood cells intimately interact with the cellular and molecular biology to regulate haemostasis and thrombosis in the context of health and disease from the macro‐ to molecular scale. We will also show how these biomechanical forces in the context of haemostasis and thrombosis have been replicated or measured in vitro.

A microengineered vascularized bleeding model that integrates the principal components of hemostasis

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an “endothelialized” microfluidic system coupled with a microengineered pneumatic valve that induces a vascular “injury”. With perfusion of whole blood, hemostatic plug formation is visualized and “in vitro bleeding time” is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.Hemostasis is a complex ensemble of events, but current bleeding assays only analyze single components like coagulation or platelet function. Here the authors present a comprehensive vascularized microfluidic mechanical injury bleeding model that addresses different aspects of the hemostatic process.

Microvasculature-on-a-chip for the long-term study of endothelial barrier dysfunction and microvascular obstruction in disease

Alterations in the mechanical properties of erythrocytes occurring in inflammatory and haematological 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 show that 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 one month enables the real-time visualization, with high spatiotemporal resolution, of microvascular obstruction and endothelial permeability under physiological flow conditions. We found that extracellular haem—a haemolytic by-product—induces delayed yet 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.An endothelialized microfluidic system that recapitulates physiological properties of the microvasculature enables the real-time visualization of vascular-pathology features associated with sickle-cell disease and malaria, with high spatiotemporal resolution.

3D microvascular model recapitulates the diffuse large B-cell lymphoma tumor microenvironment in vitro

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer that affects ∼22 000 people in the United States yearly. Understanding the complex cellular interactions of the tumor microenvironment is critical to the success and development of DLBCL treatment strategies. In vitro platforms that successfully model the complex tumor microenvironment without introducing the variability of in vivo systems are vital for understanding these interactions. To date, no such in vitro model exists that can accurately recapitulate the interactions that occur between immune cells, cancer cells, and endothelial cells in the tumor microenvironment of DLBCL. To that end, we developed a lymphoma-on-chip model consisting of a hydrogel based tumor model traversed by a vascularized, perfusable, round microchannel that successfully recapitulates key complexities and interactions of the in vivo tumor microenvironment in vitro. We have shown that the perfusion capabilities of this technique allow us to study targeted treatment strategies, as well as to model the diffusion of infused reagents spatiotemporally. Furthermore, this model employs a novel fabrication technique that utilizes common laboratory materials, and allows for the microfabrication of multiplex microvascular environments without the need for advanced microfabrication facilities. Through our facile microfabrication process, we are able to achieve micro vessels within a tumor model that are highly reliable and precise over the length of the vessel. Overall, we have developed a tool that enables researchers from many diverse disciplines to study previously inaccessible aspects of the DLBCL tumor microenvironment, with profound implications for drug delivery and design.

Platelet integrins exhibit anisotropic mechanosensing and harness piconewton forces to mediate platelet aggregation

Significance During initial growth of a clot, it is necessary for platelets to aggregate, which is mediated by binding fibrinogen molecules that bridge platelets through their integrin receptors. How platelet integrin receptors rapidly bind fibrinogen that is attached to the surface of another platelet but yet ignore soluble fibrinogen remains a mystery. This is difficult to understand since fibrinogen is the third most abundant protein in blood plasma. Here we show that differentiation between soluble and immobilized platelet ligands is mediated by mechanical forces. We demonstrate that the platelet integrins apply specific piconewton forces to test their ligands within the platelet junction. Our results provide insights into how clotting functions. Platelet aggregation at the site of vascular injury is essential in clotting. During this process, platelets are bridged by soluble fibrinogen that binds surface integrin receptors. One mystery in the mechanism of platelet aggregation pertains to how resting platelets ignore soluble fibrinogen, the third most abundant protein in the bloodstream, and yet avidly bind immobile fibrinogen on the surface of other platelets at the primary injury site. We speculate that platelet integrins are mechanosensors that test their ligands across the platelet–platelet synapse. To investigate this model, we interrogate human platelets using approaches that include the supported lipid bilayer platform as well as DNA tension sensor technologies. Experiments suggest that platelet integrins require lateral forces to mediate platelet–platelet interactions. Mechanically labile ligands dampen platelet activation, and the onset of piconewton integrin tension coincides with calcium flux. Activated platelets display immobilized fibrinogen on their surface, thus mediating further recruitment of resting platelets. The distribution of integrin tension was shown to be spatially regulated through two myosin-signaling pathways, myosin light chain kinase and Rho-associated kinase. Finally, we discovered that the termination of integrin tension is coupled with the exposure of phosphatidylserine. Our work reveals the highest spatial and temporal resolution maps of platelet integrin mechanics and its role in platelet aggregation, suggesting that platelets are physical substrates for one another that establish mechanical feedback loops of activation. The results are reminiscent of mechanical regulation of the T-cell receptor, E-cadherin, and Notch pathways, suggesting a common feature for signaling at cell junctions.

Simplified prototyping of perfusable polystyrene microfluidics

Cell culture in microfluidic systems has primarily been conducted in devices comprised of polydimethylsiloxane (PDMS) or other elastomers. As polystyrene (PS) is the most characterized and commonly used substrate material for cell culture, microfluidic cell culture would ideally be conducted in PS-based microsystems that also enable tight control of perfusion and hydrodynamic conditions, which are especially important for culture of vascular cell types. Here, we report a simple method to prototype perfusable PS microfluidics for endothelial cell culture under flow that can be fabricated using standard lithography and wet laboratory equipment to enable stable perfusion at shear stresses up to 300 dyn/cm(2) and pumping pressures up to 26 kPa for at least 100 h. This technique can also be extended to fabricate perfusable hybrid PS-PDMS microfluidics of which one application is for increased efficiency of viral transduction in non-adherent suspension cells by leveraging the high surface area to volume ratio of microfluidics and adhesion molecules that are optimized for PS substrates. These biologically compatible microfluidic devices can be made more accessible to biological-based laboratories through the outsourcing of lithography to various available microfluidic foundries.