Byungwook Ahn

Ultrasoft microgels displaying emergent platelet-like behaviours

Efforts to create platelet-like structures for the augmentation of haemostasis have focused solely on recapitulating aspects of platelet adhesion; more complex platelet behaviours such as clot contraction are assumed to be inaccessible to synthetic systems. Here, we report the creation of fully synthetic platelet-like particles (PLPs) that augment clotting in vitro under physiological flow conditions and achieve wound-triggered haemostasis and decreased bleeding times in vivo in a traumatic injury model. PLPs were synthesized by combining highly deformable microgel particles with molecular-recognition motifs identified through directed evolution. In vitro and in silico analyses demonstrate that PLPs actively collapse fibrin networks, an emergent behaviour that mimics in vivo clot contraction. Mechanistically, clot collapse is intimately linked to the unique deformability and affinity of PLPs for fibrin fibres, as evidenced by dissipative particle dynamics simulations. Our findings should inform the future design of a broader class of dynamic, biosynthetic composite materials.

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.

Anionic lipids are required for Vesicular Stomatitis Virus G protein-mediated single particle fusion with supported lipid bilayers

Background: Regulation of virus entry by host lipids is poorly understood. Results: Imaging of low pH-mediated fusion between single vesicular stomatitis pseudoviruses and lipid bilayers revealed a striking reliance on anionic lipids. Conclusion: The dependence of fusion on late endosome-resident anionic lipids suggests a new means for regulating the virus entry sites. Significance: Reliance on specific lipids for fusion may shed light on future antiviral strategies. Viral glycoproteins mediate fusion between viral and cellular membranes upon binding to cognate receptors and/or experiencing low pH. Although activation of viral glycoproteins is thought to be necessary and sufficient for fusion, accumulating evidence suggests that additional cellular factors, including lipids, can modulate the fusion process. Understanding the role of lipids in virus entry via endocytosis is impeded by poor accessibility and the highly diverse nature of endosomes. Here we imaged fusion of single retroviral particles pseudotyped with the vesicular stomatitis virus (VSV) G protein with dextran-supported lipid bilayers. Incorporation of diffusible fluorescent labels into the viral membrane and the viral interior enabled detection of the lipid mixing (hemifusion) and content transfer (full fusion) steps of VSV G-mediated fusion at low pH. Although single virus fusion with supported bilayers made of zwitterionic lipids could not be detected, inclusion of anionic lipids, phosphatidylserine, and bis(monoacylglycero)phosphate (BMP), greatly enhanced the efficiency of hemifusion and permitted full fusion. Importantly, lipid mixing always preceded the opening of a fusion pore, demonstrating that VSV G-mediated fusion proceeds through a long-lived hemifusion intermediate. Kinetic analysis of lipid and content transfer showed that the lags between lipid and content mixing defining the lifetime of a hemifusion intermediate were significantly shorter for BMP-containing compared with PS-containing bilayers. The strong fusion-enhancing effect of BMP, a late endosome-resident lipid, is consistent with the model that VSV initiates fusion in early endosomes but releases its core into the cytosol after reaching late endosomal compartments.

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales (1,2). In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions(3-6). As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions(6-13).However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature. Here, we report a method to create an endothelialized in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This microvasculature-on-a-chip provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

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.

Using microfluidics to investigate hematopoietic stem cell and microniche interactions at the single cell level.

In recent years, microfluidic devices have become widely used in biology, and with the advantage of requiring low sample volumes, enables previously technologically infeasible experiments in hematopoietic stem cell (HSC) research. Here, we introduce a microfluidic device to investigate dynamic interactions between HSC and model niches in vitro. The device comprises a pneumatic valve which enables the culturing of different types of niche cells in different parts of the same device. Single HSCs can then be injected into the microfluidic device, manipulated, and placed onto different niches within the same device as controlled by the user. Here, we describe the device fabrication method, the HSC collection methodology, and the operational procedure for the device.