Publications by authors named "David R Myers"

27 Publications

  • Page 1 of 1

Platelet heterogeneity enhances blood clot volumetric contraction: An example of asynchrono-mechanical amplification.

Biomaterials 2021 Jul 23;274:120828. Epub 2021 Apr 23.

George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, 801 Ferst Drive, Atlanta, GA, 30332-0405, USA. Electronic address:

Physiological processes such as blood clotting and wound healing as well as pathologies such as fibroses and musculoskeletal contractures, all involve biological materials composed of a contracting cellular population within a fibrous matrix, yet how the microscale interactions among the cells and the matrix lead to the resultant emergent behavior at the macroscale tissue level remains poorly understood. Platelets, the anucleate cell fragments that do not divide nor synthesize extracellular matrix, represent an ideal model to study such systems. During blood clot contraction, microscopic platelets actively pull fibers to shrink the macroscale clot to less than 10% of its initial volume. We discovered that platelets utilize a new emergent behavior, asynchrono-mechanical amplification, to enhanced volumetric material contraction and to magnify contractile forces. This behavior is triggered by the heterogeneity in the timing of a population of actuators. This result indicates that cell heterogeneity, often attributed to stochastic cell-to-cell variability, can carry an essential biophysical function, thereby highlighting the importance of considering 4 dimensions (space + time) in cell-matrix biomaterials. This concept of amplification via heterogeneity can be harnessed to increase mechanical efficiency in diverse systems including implantable biomaterials, swarm robotics, and active polymer composites.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184644PMC
July 2021

Vascularized Microfluidics and Their Untapped Potential for Discovery in Diseases of the Microvasculature.

Annu Rev Biomed Eng 2021 Apr 16. Epub 2021 Apr 16.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332, USA; email:

Microengineering advances have enabled the development of perfusable, endothelialized models of the microvasculature that recapitulate the unique biological and biophysical conditions of the microcirculation in vivo. Indeed, at that size scale (<100 μm)-where blood no longer behaves as a simple continuum fluid; blood cells approximate the size of the vessels themselves; and complex interactions among blood cells, plasma molecules, and the endothelium constantly ensue-vascularized microfluidics are ideal tools to investigate these microvascular phenomena. Moreover, perfusable, endothelialized microfluidics offer unique opportunities for investigating microvascular diseases by enabling systematic dissection of both the blood and vascular components of the pathophysiology at hand. We review () the state of the art in microvascular devices and () the myriad of microvascular diseases and pressing challenges. The engineering community has unique opportunities to innovate with new microvascular devices and to partner with biomedical researchers to usher in a new era of understanding and discovery of microvascular diseases. Expected final online publication date for the , Volume 23 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
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http://dx.doi.org/10.1146/annurev-bioeng-091520-025358DOI Listing
April 2021

Label-free hematology analysis using deep-ultraviolet microscopy.

Proc Natl Acad Sci U S A 2020 06 19;117(26):14779-14789. Epub 2020 Jun 19.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332;

Hematological analysis, via a complete blood count (CBC) and microscopy, is critical for screening, diagnosing, and monitoring blood conditions and diseases but requires complex equipment, multiple chemical reagents, laborious system calibration and procedures, and highly trained personnel for operation. Here we introduce a hematological assay based on label-free molecular imaging with deep-ultraviolet microscopy that can provide fast quantitative information of key hematological parameters to facilitate and improve hematological analysis. We demonstrate that this label-free approach yields 1) a quantitative five-part white blood cell differential, 2) quantitative red blood cell and hemoglobin characterization, 3) clear identification of platelets, and 4) detailed subcellular morphology. Analysis of tens of thousands of live cells is achieved in minutes without any sample preparation. Finally, we introduce a pseudocolorization scheme that accurately recapitulates the appearance of cells under conventional staining protocols for microscopic analysis of blood smears and bone marrow aspirates. Diagnostic efficacy is evaluated by a panel of hematologists performing a blind analysis of blood smears from healthy donors and thrombocytopenic and sickle cell disease patients. This work has significant implications toward simplifying and improving CBC and blood smear analysis, which is currently performed manually via bright-field microscopy, and toward the development of a low-cost, easy-to-use, and fast hematological analyzer as a point-of-care device and for low-resource settings.
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http://dx.doi.org/10.1073/pnas.2001404117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334528PMC
June 2020

The biophysics and mechanics of blood from a materials perspective.

Nat Rev Mater 2019 May 28;4(5):294-311. Epub 2019 Mar 28.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.

Cells actively interact with their microenvironment, constantly sensing and modulating biochemical and biophysical signals. Blood comprises a variety of non-adherent cells that interact with each other and with endothelial and vascular smooth muscle cells of the blood vessel walls. Blood cells are further experiencing a range of external forces by the hemodynamic environment and they also exert forces to remodel their local environment. Therefore, the biophysics and material properties of blood cells and blood play an important role in determining blood behaviour in health and disease. In this Review, we discuss blood cells and tissues from a materials perspective, considering the mechanical properties and biophysics of individual blood cells and endothelial cells as well as blood cell collectives. We highlight how blood vessels provide a mechanosensitive barrier between blood and tissues and how changes in vessel stiffness and flow shear stress can be correlated to plaque formation and exploited for the design of vascular grafts. We discuss the effect of the properties of fibrin on blood clotting, and investigate how forces exerted by platelets are correlated to disease. Finally, we hypothesize that blood and vascular cells are constantly establishing a mechanical homeostasis, which, when imbalanced, can lead to hematologic and vascular diseases.
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http://dx.doi.org/10.1038/s41578-019-0099-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238390PMC
May 2019

Getting a good view: imaging of platelets under flow.

Platelets 2020 Jul 28;31(5):570-579. Epub 2020 Feb 28.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University , Atlanta, GA, USA.

As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our imaging studies of platelets under flow.
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http://dx.doi.org/10.1080/09537104.2020.1732320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332395PMC
July 2020

Feeling the Force: Measurements of Platelet Contraction and Their Diagnostic Implications.

Semin Thromb Hemost 2019 Apr 19;45(3):285-296. Epub 2018 Dec 19.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia.

In addition to the classical biological and biochemical framework, blood clots can also be considered as active biomaterials composed of dynamically contracting platelets, nascent polymeric fibrin that functions as a matrix scaffold, and entrapped blood cells. As platelets sense, rearrange, and apply forces to the surrounding microenvironment, they dramatically change the material properties of the nascent clot, increasing its stiffness by an order of magnitude. Hence, the mechanical properties of blood clots are intricately tied to the forces applied by individual platelets. Research has also shown that the pathophysiological changes in clot mechanical properties are associated with bleeding and clotting disorders, cancer, stroke, ischemic heart disease, and more. By approaching the study of hemostasis and thrombosis from a biophysical and mechanical perspective, important insights have been made into how the mechanics of clotting and the forces applied by platelets are linked to various diseases. This review will familiarize the reader with a mechanics framework that is contextualized with relevant biology. The review also includes a discussion of relevant tools used to study platelet forces either directly or indirectly, and finally, concludes with a summary of potential links between clotting forces and disease.
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http://dx.doi.org/10.1055/s-0038-1676315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284283PMC
April 2019

Smartphone app for non-invasive detection of anemia using only patient-sourced photos.

Nat Commun 2018 12 4;9(1):4924. Epub 2018 Dec 4.

The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, GA, USA.

We introduce a paradigm of completely non-invasive, on-demand diagnostics that may replace common blood-based laboratory tests using only a smartphone app and photos. We initially targeted anemia, a blood condition characterized by low blood hemoglobin levels that afflicts >2 billion people. Our app estimates hemoglobin levels by analyzing color and metadata of fingernail bed smartphone photos and detects anemia (hemoglobin levels <12.5 g dL) with an accuracy of ±2.4 g dL and a sensitivity of 97% (95% CI, 89-100%) when compared with CBC hemoglobin levels (n = 100 subjects), indicating its viability to serve as a non-invasive anemia screening tool. Moreover, with personalized calibration, this system achieves an accuracy of ±0.92 g dL of CBC hemoglobin levels (n = 16), empowering chronic anemia patients to serially monitor their hemoglobin levels instantaneously and remotely. Our on-demand system enables anyone with a smartphone to download an app and immediately detect anemia anywhere and anytime.
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http://dx.doi.org/10.1038/s41467-018-07262-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279826PMC
December 2018

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

Nat Commun 2018 02 6;9(1):509. Epub 2018 Feb 6.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 345 Ferst Drive, Atlanta, GA, 30332, USA.

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.
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http://dx.doi.org/10.1038/s41467-018-02990-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802762PMC
February 2018

Towards remote assessment and screening of acute abdominal pain using only a smartphone with native accelerometers.

Sci Rep 2017 10 6;7(1):12750. Epub 2017 Oct 6.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA, 30332, USA.

Smartphone-based telehealth holds the promise of shifting healthcare from the clinic to the home, but the inability for clinicians to conduct remote palpation, or touching, a key component of the physical exam, remains a major limitation. This is exemplified in the assessment of acute abdominal pain, in which a physician's palpation determines if a patient's pain is life-threatening requiring emergency intervention/surgery or due to some less-urgent cause. In a step towards virtual physical examinations, we developed and report for the first time a "touch-capable" mHealth technology that enables a patient's own hands to serve as remote surrogates for the physician's in the screening of acute abdominal pain. Leveraging only a smartphone with its native accelerometers, our system guides a patient through an exact probing motion that precisely matches the palpation motion set by the physician. An integrated feedback algorithm, with 95% sensitivity and specificity, enabled 81% of tested patients to match a physician abdominal palpation curve with <20% error after 6 attempts. Overall, this work addresses a key issue in telehealth that will vastly improve its capabilities and adoption worldwide.
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http://dx.doi.org/10.1038/s41598-017-13076-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630621PMC
October 2017

Microfluidic Transduction Harnesses Mass Transport Principles to Enhance Gene Transfer Efficiency.

Mol Ther 2017 10 8;25(10):2372-2382. Epub 2017 Jul 8.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322, USA; Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. Electronic address:

Ex vivo gene therapy using lentiviral vectors (LVs) is a proven approach to treat and potentially cure many hematologic disorders and malignancies but remains stymied by cumbersome, cost-prohibitive, and scale-limited production processes that cannot meet the demands of current clinical protocols for widespread clinical utilization. However, limitations in LV manufacture coupled with inefficient transduction protocols requiring significant excess amounts of vector currently limit widespread implementation. Herein, we describe a microfluidic, mass transport-based approach that overcomes the diffusion limitations of current transduction platforms to enhance LV gene transfer kinetics and efficiency. This novel ex vivo LV transduction platform is flexible in design, easy to use, scalable, and compatible with standard cell transduction reagents and LV preparations. Using hematopoietic cell lines, primary human T cells, primary hematopoietic stem and progenitor cells (HSPCs) of both murine (Sca-1) and human (CD34) origin, microfluidic transduction using clinically processed LVs occurs up to 5-fold faster and requires as little as one-twentieth of LV. As an in vivo validation of the microfluidic-based transduction technology, HSPC gene therapy was performed in hemophilia A mice using limiting amounts of LV. Compared to the standard static well-based transduction protocols, only animals transplanted with microfluidic-transduced cells displayed clotting levels restored to normal.
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http://dx.doi.org/10.1016/j.ymthe.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5628863PMC
October 2017

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

Nat Commun 2017 06 8;8:15594. Epub 2017 Jun 8.

Department of Bioengineering, Rice University, Houston, Texas 77005, USA.

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.
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http://dx.doi.org/10.1038/ncomms15594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472756PMC
June 2017

Platelet-Microcapsule Hybrids Leverage Contractile Force for Targeted Delivery of Hemostatic Agents.

ACS Nano 2017 06 5;11(6):5579-5589. Epub 2017 Jun 5.

Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta/Emory University School of Medicine , Atlanta, Georgia 30322, United States.

We report a cell-mediated, targeted drug delivery system utilizing polyelectrolyte multilayer capsules that hybridize with the patient's own platelets upon intravenous administration. The hybridized platelets function as the sensor and actuator for targeted drug delivery and controlled release in our system. These capsules are biochemically and mechanically tuned to enable platelet adhesion and capsule rupture upon platelet activation and contraction, enabling the targeted and controlled "burst" release of an encapsulated biotherapeutic. As platelets are the "first responders" in the blood clot formation process, this platelet-hybridized system is ideal for the targeted delivery of clot-augmenting biotherapeutics wherein immediate therapeutic efficacy is required. As proof-of-concept, we tailored this system to deliver the pro-clotting biotherapeutic factor VIII for hemophilia A patients that have developed inhibitory antifactor VIII antibodies. The polyelectrolyte multilayer capsules physically shield the encapsulated factor VIII from the patient's inhibitors during circulation, preserving its bioactivity until it is delivered at the target site via platelet contractile force. Using an in vitro microfluidic vascular injury model with factor VIII-inhibited blood, we demonstrate a 3.8× increase in induced fibrin formation using capsules loaded with factor VIII at a concentration an order of magnitude lower than that used in systemic delivery. We further demonstrate that clot formation occurs 18 min faster when factor VIII loaded capsules are used compared to systemic delivery at the same concentration. Because platelets are integral in the pathophysiology of thrombotic disorders, cancer, and innate immunity, this paradigm-shifting smart drug delivery system can be similarly applied to these diseases.
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http://dx.doi.org/10.1021/acsnano.7b00929DOI Listing
June 2017

Single-platelet nanomechanics measured by high-throughput cytometry.

Nat Mater 2017 02 10;16(2):230-235. Epub 2016 Oct 10.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

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 drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. 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 and thrombotic disorders. 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.
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http://dx.doi.org/10.1038/nmat4772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266633PMC
February 2017

Cellular softening mediates leukocyte demargination and trafficking, thereby increasing clinical blood counts.

Proc Natl Acad Sci U S A 2016 Feb 8;113(8):1987-92. Epub 2016 Feb 8.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA 30332; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322; Winship Cancer Institute, Emory University, Atlanta, GA 30322; Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332; Institute for Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA 30332;

Leukocytes normally marginate toward the vascular wall in large vessels and within the microvasculature. Reversal of this process, leukocyte demargination, leads to substantial increases in the clinical white blood cell and granulocyte count and is a well-documented effect of glucocorticoid and catecholamine hormones, although the underlying mechanisms remain unclear. Here we show that alterations in granulocyte mechanical properties are the driving force behind glucocorticoid- and catecholamine-induced demargination. First, we found that the proportions of granulocytes from healthy human subjects that traversed and demarginated from microfluidic models of capillary beds and veins, respectively, increased after the subjects ingested glucocorticoids. Also, we show that glucocorticoid and catecholamine exposure reorganizes cellular cortical actin, significantly reducing granulocyte stiffness, as measured with atomic force microscopy. Furthermore, using simple kinetic theory computational modeling, we found that this reduction in stiffness alone is sufficient to cause granulocyte demargination. Taken together, our findings reveal a biomechanical answer to an old hematologic question regarding how glucocorticoids and catecholamines cause leukocyte demargination. In addition, in a broader sense, we have discovered a temporally and energetically efficient mechanism in which the innate immune system can simply alter leukocyte stiffness to fine tune margination/demargination and therefore leukocyte trafficking in general. These observations have broad clinically relevant implications for the inflammatory process overall as well as hematopoietic stem cell mobilization and homing.
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http://dx.doi.org/10.1073/pnas.1508920113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776450PMC
February 2016

"Do-it-yourself in vitro vasculature that recapitulates in vivo geometries for investigating endothelial-blood cell interactions".

Sci Rep 2015 Jul 23;5:12401. Epub 2015 Jul 23.

1] Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University [2] Emory University School of Medicine, Department of Pediatrics, Division of Pediatric Hematology/Oncology [3] Childrens Healthcare of Atlanta, Aflac Cancer &Blood Disorders Center [4] Institute of Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA, United States.

Investigating biophysical cellular interactions in the circulation currently requires choosing between in vivo models, which are difficult to interpret due in part to the hemodynamic and geometric complexities of the vasculature; or in vitro systems, which suffer from non-physiologic assumptions and/or require specialized microfabrication facilities and expertise. To bridge that gap, we developed an in vitro "do-it-yourself" perfusable vasculature model that recapitulates in vivo geometries, such as aneurysms, stenoses, and bifurcations, and supports endothelial cell culture. These inexpensive, disposable devices can be created rapidly (<2 hours) with high precision and repeatability, using standard off-the-shelf laboratory supplies. Using these "endothelialized" systems, we demonstrate that spatial variation in vascular cell adhesion molecule (VCAM-1) expression correlates with the wall shear stress patterns of vascular geometries. We further observe that the presence of endothelial cells in stenoses reduces platelet adhesion but increases sickle cell disease (SCD) red blood cell (RBC) adhesion in bifurcations. Overall, our method enables researchers from all disciplines to study cellular interactions in physiologically relevant, yet simple-to-make, in vitro vasculature models.
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http://dx.doi.org/10.1038/srep12401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4894411PMC
July 2015

Platelets and physics: How platelets "feel" and respond to their mechanical microenvironment.

Blood Rev 2015 Nov 8;29(6):377-86. Epub 2015 May 8.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University, School of Medicine, Atlanta, GA 30322, USA; Winship Cancer Institute of Emory University, Atlanta, GA 30322, USA; Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.blre.2015.05.002DOI Listing
November 2015

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition.

Blood 2015 Jul 11;126(4):531-8. Epub 2015 May 11.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Atlanta, GA; Department of Pediatrics, Emory University School of Medicine, Atlanta, GA; Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA; Winship Cancer Institute, Emory University, Atlanta, GA; Institute of Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA;

Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet α-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-µm features. Interestingly, α-granule exocytosis and deposition of the α-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and αIIbβ3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack α-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium.
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http://dx.doi.org/10.1182/blood-2014-11-607614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513253PMC
July 2015

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.

Blood 2015 Aug 30;126(6):817-24. Epub 2015 Apr 30.

Wallace C Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA; Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Children's Healthcare of Atlanta and Emory University School of Medicine, Atlanta, GA; Institute of Electronics and Nanotechnology, Georgia Institute of Technology, Atlanta, GA;

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model.
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http://dx.doi.org/10.1182/blood-2015-02-628594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528067PMC
August 2015

Platelet mechanosensing of collagen matrices.

PLoS One 2015 27;10(4):e0126624. Epub 2015 Apr 27.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, United States of America; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia, United States of America; Winship Cancer Institute of Emory University, Atlanta, Georgia, United States of America.

During vascular injury, platelets adhere to exposed subendothelial proteins, such as collagen, on the blood vessel walls to trigger clot formation. Although the biochemical signalings of platelet-collagen interactions have been well characterized, little is known about the role microenvironmental biomechanical properties, such as vascular wall stiffness, may have on clot formation. To that end, we investigated how substrates of varying stiffness conjugated with the same concentration of Type I collagen affect platelet adhesion, spreading, and activation. Using collagen-conjugated polyacrylamide (PA) gels of different stiffnesses, we observed that platelets do in fact mechanotransduce the stiffness cues of collagen substrates, manifesting in increased platelet spreading on stiffer substrates. In addition, increasing substrate stiffness also increases phosphatidylserine exposure, a key aspect of platelet activation that initiates coagulation on the platelet surface. Mechanistically, these collagen substrate stiffness effects are mediated by extracellular calcium levels and actomyosin pathways driven by myosin light chain kinase but not Rho-associated protein kinase. Overall, our results improve our understanding of how the mechanics of different tissues and stroma affect clot formation, what role the increased vessel wall stiffness in atherosclerosis may directly have on thrombosis leading to heart attacks and strokes, and how age-related increased vessel wall stiffness affects hemostasis and thrombosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0126624PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4411076PMC
April 2016

Simplified prototyping of perfusable polystyrene microfluidics.

Biomicrofluidics 2014 Jul 30;8(4):046501. Epub 2014 Jul 30.

The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta , Georgia 30332, USA.

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.
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http://dx.doi.org/10.1063/1.4892035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4189295PMC
July 2014

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

Proc Natl Acad Sci U S A 2014 Oct 22;111(40):14430-5. Epub 2014 Sep 22.

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332; Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA 30322; Winship Cancer Institute of Emory University, Atlanta, GA 30322; Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332

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.
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http://dx.doi.org/10.1073/pnas.1322917111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210024PMC
October 2014

Mitochondrially mediated integrin αIIbβ3 protein inactivation limits thrombus growth.

J Biol Chem 2013 Oct 6;288(42):30672-30681. Epub 2013 Sep 6.

From the Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322,; the Aflac Cancer and Blood Disorders Center, Children's Healthcare of Atlanta, Atlanta, Georgia 30322. Electronic address:

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closely with integrin αIIbβ3 inactivation. Calpain inhibition blocked integrin β3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.
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http://dx.doi.org/10.1074/jbc.M113.472688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798537PMC
October 2013

Loss of the F-BAR protein CIP4 reduces platelet production by impairing membrane-cytoskeleton remodeling.

Blood 2013 Sep 23;122(10):1695-706. Epub 2013 Jul 23.

Department of Pediatrics, Robert H. Lurie Comprehensive Cancer Center and Children's Memorial Hospital, Northwestern Feinberg School of Medicine, Chicago, IL 60611, USA.

Megakaryocytes generate platelets through extensive reorganization of the cytoskeleton and plasma membrane. Cdc42 interacting protein 4 (CIP4) is an F-BAR protein that localizes to membrane phospholipids through its BAR domain and interacts with Wiskott-Aldrich Syndrome Protein (WASP) via its SRC homology 3 domain. F-BAR proteins promote actin polymerization and membrane tubulation. To study its function, we generated CIP4-null mice that displayed thrombocytopenia similar to that of WAS(-) mice. The number of megakaryocytes and their progenitors was not affected. However, the number of proplatelet protrusions was reduced in CIP4-null, but not WAS(-), megakaryocytes. Electron micrographs of CIP4-null megakaryocytes showed an altered demarcation membrane system. Silencing of CIP4, not WASP, expression resulted in fewer proplatelet-like extensions. Fluorescence anisotropy studies showed that loss of CIP4 resulted in a more rigid membrane. Micropipette aspiration demonstrated decreased cortical actin tension in megakaryocytic cells with reduced CIP4 or WASP protein. These studies support a new biophysical mechanism for platelet biogenesis whereby CIP4 enhances the complex, dynamic reorganization of the plasma membrane (WASP independent) and actin cortex network (as known for WASP and cortical actin) to reduce the work required for generating proplatelets. CIP4 is a new component in the highly coordinated system of megakaryocytic membrane and cytoskeletal remodeling affecting platelet production.
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http://dx.doi.org/10.1182/blood-2013-03-484550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3765055PMC
September 2013

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

J Cell Mol Med 2013 May 14;17(5):579-96. Epub 2013 Mar 14.

Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

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.
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http://dx.doi.org/10.1111/jcmm.12041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822810PMC
May 2013

Osmosis is not driven by water dilution.

Trends Plant Sci 2013 Apr 5;18(4):195-7. Epub 2013 Jan 5.

Department of Physics, Bard College at Simon's Rock, Great Barrington, MA 01201, USA.

There is a misconception among plant scientists that osmosis is driven by the tendency of solutes to dilute water. In this opinion article, we discuss the quantitative and qualitative failures of this view, and go on to review the correct kinetic picture of osmosis as it appears in physics textbooks.
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http://dx.doi.org/10.1016/j.tplants.2012.12.001DOI Listing
April 2013

Endothelialized microfluidics for studying microvascular interactions in hematologic diseases.

J Vis Exp 2012 Jun 22(64). Epub 2012 Jun 22.

Department of Pediatrics, Emory University School of Medicine, Georgia, USA. .

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.
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http://dx.doi.org/10.3791/3958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471282PMC
June 2012

Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level.

PLoS One 2011 20;6(10):e26437. Epub 2011 Oct 20.

Division of Pediatric Hematology/Oncology, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, United States of America.

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 µm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 µm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026437PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197646PMC
February 2012