Publications by authors named "Keith B Neeves"

55 Publications

The Art and Science of Building a Computational Model to Understand Hemostasis.

Semin Thromb Hemost 2021 Mar 26;47(2):129-138. Epub 2021 Feb 26.

Department of Bioengineering, Department of Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplant, Hemophilia and Thrombosis Center, University of Colorado, Denver, Colorado.

Computational models of various facets of hemostasis and thrombosis have increased substantially in the last decade. These models have the potential to make predictions that can uncover new mechanisms within the complex dynamics of thrombus formation. However, these predictions are only as good as the data and assumptions they are built upon, and therefore model building requires intimate coupling with experiments. The objective of this article is to guide the reader through how a computational model is built and how it can inform and be refined by experiments. This is accomplished by answering six questions facing the model builder: (1) Why make a model? (2) What kind of model should be built? (3) How is the model built? (4) Is the model a "good" model? (5) Do we believe the model? (6) Is the model useful? These questions are answered in the context of a model of thrombus formation that has been successfully applied to understanding the interplay between blood flow, platelet deposition, and coagulation and in identifying potential modifiers of thrombin generation in hemophilia A.
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http://dx.doi.org/10.1055/s-0041-1722861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7920145PMC
March 2021

Computationally Driven Discovery in Coagulation.

Arterioscler Thromb Vasc Biol 2021 01 29;41(1):79-86. Epub 2020 Oct 29.

Department of Applied Mathematics and Statistics, Colorado School of Mines, Golden (K.L.).

Bleeding frequency and severity within clinical categories of hemophilia A are highly variable and the origin of this variation is unknown. Solving this mystery in coagulation requires the generation and analysis of large data sets comprised of experimental outputs or patient samples, both of which are subject to limited availability. In this review, we describe how a computationally driven approach bypasses such limitations by generating large synthetic patient data sets. These data sets were created with a mechanistic mathematical model, by varying the model inputs, clotting factor, and inhibitor concentrations, within normal physiological ranges. Specific mathematical metrics were chosen from the model output, used as a surrogate measure for bleeding severity, and statistically analyzed for further exploration and hypothesis generation. We highlight results from our recent study that employed this computationally driven approach to identify FV (factor V) as a key modifier of thrombin generation in mild to moderate hemophilia A, which was confirmed with complementary experimental assays. The mathematical model was used further to propose a potential mechanism for these observations whereby thrombin generation is rescued in FVIII-deficient plasma due to reduced substrate competition between FV and FVIII for FXa (activated factor X).
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http://dx.doi.org/10.1161/ATVBAHA.120.314648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769924PMC
January 2021

Transport of Colloidal Particles in Microscopic Porous Medium Analogues with Surface Charge Heterogeneity: Experiments and the Fundamental Role of Single-Bead Deposition.

Environ Sci Technol 2020 11 20;54(21):13651-13660. Epub 2020 Oct 20.

Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado 80401, United States.

Understanding colloid transport in subsurface environments is challenging because of complex interactions among colloids, groundwater, and porous media over several length scales. Here, we report a versatile method to assemble bead-based microfluidic porous media analogues with chemical heterogeneities of different configurations. We further study the transport of colloidal particles through a family of porous media analogues that are randomly packed with oppositely charged beads with different mixing ratios. We recorded the dynamics of colloidal particle deposition at the level of single grains. From these, the maximum surface coverage (θ = 0.051) was measured directly. The surface-blocking function and the deposition coefficient ( = 3.56 s) were obtained. Using these pore-scale parameters, the transport of colloidal particles was modeled using a one-dimensional advection-dispersion-deposition equation under the assumption of irreversible adsorption between oppositely charged beads and colloids, showing very good agreement with experimental breakthrough curves and retention profiles at the scale of the entire porous medium analogue. This work presents a new approach to fabricate chemically heterogeneous porous media in a microfluidic device that enables the direct measurement of pore-scale colloidal deposition. Compared with the conventional curve-fitting method for deposition constant, our approach allows quantitative prediction of colloidal breakthrough and retention coupling of direct pore-scale measurements and an advection-dispersion-deposition model.
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http://dx.doi.org/10.1021/acs.est.0c03225DOI Listing
November 2020

Pathologic Shear and Elongation Rates Do Not Cause Cleavage of Von Willebrand Factor by ADAMTS13 in a Purified System.

Cell Mol Bioeng 2020 Aug 17;13(4):379-390. Epub 2020 Jul 17.

Department of Pediatrics, Washington University in St. Louis, 660 S. Euclid Avenue, Campus Box 8208, 5th floor MPRB, St. Louis, MO 63110 USA.

Introduction: Pathological flows in patients with severe aortic stenosis are associated with acquired von Willebrand syndrome. This syndrome is characterized by excessive cleavage of von Willebrand factor by its main protease, A Disintegrin and Metalloproteinase with a Thrombospondin Type 1 Motif, Member 13 (ADAMTS13) leading to decreased VWF function and mucocutaneous bleeding. Aortic valve replacement and correction of the flow behavior to physiological levels reverses the syndrome, supporting the association between pathological flow and acquired von Willebrand syndrome. We investigated the effects of shear and elongational rates on von Willebrand factor cleavage in the presence of ADAMTS13.

Methods: We identified acquired von Willebrand syndrome in five patients with severe aortic stenosis. Doppler echography values from these patients were used to develop three computational fluid dynamic (CFD) aortic valve models (normal, mild and severe stenosis). Shear, elongational rates and exposure times identified in the CFD simulations were used as parameters for the design of microfluidic devices to test the effects of pathologic shear and elongational rates on the structure and function of von Willebrand factor.

Results: The shear rates (0-10,000s), elongational rates (0-1000 s) and exposure times (1-180 ms) tested in our microfluidic designs mimicked the flow features identified in patients with aortic stenosis. The shear and elongational rates tested did not lead to excessive cleavage or decreased function of von Willebrand factor in the presence of the protease.

Conclusions: High shear and elongational rates in the presence of ADAMTS13 are not sufficient for excessive cleavage of von Willebrand Factor.
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http://dx.doi.org/10.1007/s12195-020-00631-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7479076PMC
August 2020

Adsorption and Absorption of Collagen Peptides to Polydimethlysiloxane and Its Influence on Platelet Adhesion Flow Assays.

Micromachines (Basel) 2020 Jan 5;11(1). Epub 2020 Jan 5.

Departments of Bioengineering and Pediatrics, University of Colorado Denver|Anschutz Medical Campus, Aurora, CO 80045, USA.

Collagen peptides are an alternative to animal derived collagens for platelet function studies under flow. The purpose of this study was to examine the use of collagen peptides in polydimethylsiloxane (PDMS) devices. Three collagen peptides with amino acid sequences and structures that capture von Willebrand factor and bind it with the platelet receptors integrin αβ and glycoprotein VI were patterned on glass, silicon, and PDMS. Each of these surfaces was also functionalized with tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane (FOTS). Surfaces were characterized by their ability to support platelet adhesion, topology by atomic force microscopy, contact angle, and peptides absorption. PDMS readily absorbs collagen peptides, depleting them from solution, thus reducing their adsorption to glass and silicon substrates when used for micropatterning. Treatment of PDMS with FOTS, but not bovine serum albumin or poloxamer 407, inhibits collagen peptide absorption and supports adsorption and platelet adhesion at venous and arterial shear rates. Similarly, FOTS treatment of glass or silicon supports collagen peptide adsorption even in the presence of untreated PDMS. In conclusion, PDMS acts as an absorptive sink for collagen peptides, rendering a non-adhesive surface for platelet adhesion and competing for peptides when used for micropatterning. The absorption of collagen peptides can be overcome by functionalization of PDMS with a fluorinated alkyl silane, thus allowing its use as a material for micropatterning or as a surface for platelet adhesion flow assays.
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http://dx.doi.org/10.3390/mi11010062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019490PMC
January 2020

A MATHEMATICAL MODEL OF PLATELET AGGREGATION IN AN EXTRAVASCULAR INJURY UNDER FLOW.

Multiscale Model Simul 2020 18;18(4):1489-1524. Epub 2020 Nov 18.

Department of Mathematics, University of California, Davis, Davis, CA 95616 USA.

We present the first mathematical model of flow-mediated primary hemostasis in an extravascular injury which can track the process from initial deposition to occlusion. The model consists of a system of ordinary differential equations (ODEs) that describe platelet aggregation (adhesion and cohesion), soluble-agonist-dependent platelet activation, and the flow of blood through the injury. The formation of platelet aggregates increases resistance to flow through the injury, which is modeled using the Stokes-Brinkman equations. Data from analogous experimental (microfluidic flow) and partial differential equation models informed parameter values used in the ODE model description of platelet adhesion, cohesion, and activation. This model predicts injury occlusion under a range of flow and platelet activation conditions. Simulations testing the effects of shear and activation rates resulted in delayed occlusion and aggregate heterogeneity. These results validate our hypothesis that flow-mediated dilution of activating chemical adenosine diphosphate hinders aggregate development. This novel modeling framework can be extended to include more mechanisms of platelet activation as well as the addition of the biochemical reactions of coagulation, resulting in a computationally efficient high throughput screening tool of primary and secondary hemostasis.
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http://dx.doi.org/10.1137/20m1317785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051825PMC
November 2020

Microwheels on Microroads: Enhanced Translation on Topographic Surfaces.

Sci Robot 2019 07;4(32)

Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado, USA 80401.

Microbot locomotion is challenging because of the reversible nature of microscale fluid flow, a limitation that can be overcome by breaking flowfield symmetry with a nearby surface. We have used this strategy with rotating wheel-shaped microbots, μwheels, that roll on surfaces leading to enhanced propulsion and fast translation speeds. Despite this, studies to date on flat surfaces show that μwheels roll inefficiently with significant slip. Taking inspiration from the mathematics of roads and wheels, here we demonstrate that μwheel velocities can be significantly enhanced by changing microroad topography. In this, we observe that periodic bumps in the road can be used to enhance the traction between μwheels and nearby walls. While continuous μwheel rotation with slip is observed on flat surfaces, a combination of rotation with slip and non-slip flip occurs when μwheels roll upon surfaces with periodic features, resulting in up to four-fold enhancement in translation velocity. The surprisingly fast rolling speed of μwheels on bumpy roads can be attributed to the hydrodynamic coupling between μwheels and road surface features, allowing non-slip rotation of entire wheels along one of their stationary edges. This road/wheel coupling can also be used to enhance μwheel sorting and separation where the gravitational potential energy barrier induced by topographic surfaces can lead to motion in only one direction and to different rolling speeds between isomeric wheels, allowing one to separate them not based on size but on symmetry.
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http://dx.doi.org/10.1126/scirobotics.aaw9525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779173PMC
July 2019

Tranexamic acid for trauma: Repackaged and redelivered.

J Thromb Haemost 2019 10;17(10):1626-1628

Departments of Bioengineering and Pediatrics, Hemophilia and Thrombosis Center, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO.

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http://dx.doi.org/10.1111/jth.14608DOI Listing
October 2019

A mathematical model of coagulation under flow identifies factor V as a modifier of thrombin generation in hemophilia A.

J Thromb Haemost 2020 02 1;18(2):306-317. Epub 2019 Nov 1.

Department of Bioengineering, University of Colorado, Aurora, CO, USA.

Background: The variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown.

Objective: To use a previously validated mathematical model of flow-mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency.

Methods: We performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen-tissue factor (TF) surfaces at a shear rate of 100 per second.

Results: Simulations identified low-normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high-normal prothrombin (150%). Low-normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high-normal prothrombin levels in both experimental models.

Conclusions: These results show that low-normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII-deficient blood.
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http://dx.doi.org/10.1111/jth.14653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994344PMC
February 2020

Engineered microparticles and nanoparticles for fibrinolysis.

J Thromb Haemost 2019 12 7;17(12):2004-2015. Epub 2019 Oct 7.

Departments of Bioengineering and Pediatrics, Hemophilia and Thrombosis Center, University of Colorado Denver, Aurora, Colorado.

Fibrinolytic agents including plasmin and plasminogen activators improve outcomes in acute ischemic stroke and thrombosis by recanalizing occluded vessels. In the decades since their introduction into clinical practice, several limitations of have been identified in terms of both efficacy and bleeding risk associated with these agents. Engineered nanoparticles and microparticles address some of these limitations by improving circulation time, reducing inhibition and degradation in circulation, accelerating recanalization, improving targeting to thrombotic occlusions, and reducing off-target effects; however, many particle-based approaches have only been used in preclinical studies to date. This review covers four advances in coupling fibrinolytic agents with engineered particles: (a) modifications of plasminogen activators with macromolecules, (b) encapsulation of plasminogen activators and plasmin in polymer and liposomal particles, (c) triggered release of encapsulated fibrinolytic agents and mechanical disruption of clots with ultrasound, and (d) enhancing targeting with magnetic particles and magnetic fields. Technical challenges for the translation of these approaches to the clinic are discussed.
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http://dx.doi.org/10.1111/jth.14637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893081PMC
December 2019

Turbulent Flow Promotes Cleavage of VWF (von Willebrand Factor) by ADAMTS13 (A Disintegrin and Metalloproteinase With a Thrombospondin Type-1 Motif, Member 13).

Arterioscler Thromb Vasc Biol 2019 09 11;39(9):1831-1842. Epub 2019 Jul 11.

From the Department of Pediatrics (M.B., K.A., F.W., K.B.N., D.B., J.D.P.), University of Colorado Anschutz Medical Campus, Aurora.

Objective- Acquired von Willebrand syndrome is defined by excessive cleavage of the VWF (von Willebrand Factor) and is associated with impaired primary hemostasis and severe bleeding. It often develops when blood is exposed to nonphysiological flow such as in aortic stenosis or mechanical circulatory support. We evaluated the role of laminar, transitional, and turbulent flow on VWF cleavage and the effects on VWF function. Approach and Results- We used a vane rheometer to generate laminar, transitional, and turbulent flow and evaluate the effect of each on VWF cleavage in the presence of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13). We performed functional assays to evaluate the effect of these flows on VWF structure and function. Computational fluid dynamics was used to estimate the flow fields and forces within the vane rheometer under each flow condition. Turbulent flow is required for excessive cleavage of VWF in an ADAMTS13-dependent manner. The assay was repeated with whole blood, and the turbulent flow had the same effect. Our computational fluid dynamics results show that under turbulent conditions, the Kolmogorov scale approaches the size of VWF. Finally, cleavage of VWF in this study has functional consequences under flow as the resulting VWF has decreased ability to bind platelets and collagen. Conclusions- Turbulent flow mediates VWF cleavage in the presence of ADAMTS13, decreasing the ability of VWF to sustain platelet adhesion. These findings impact the design of mechanical circulatory support devices and are relevant to pathological environments where turbulence is added to circulation.
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http://dx.doi.org/10.1161/ATVBAHA.119.312814DOI Listing
September 2019

ac/dc Magnetic Fields for Enhanced Translation of Colloidal Microwheels.

Langmuir 2019 Mar 19;35(9):3455-3460. Epub 2019 Feb 19.

Department of Chemical and Biological Engineering , Colorado School of Mines , Golden , Colorado 80401 , United States.

Microscale devices must overcome fluid reversibility to propel themselves in environments where viscous forces dominate. One approach, used by colloidal microwheels (μwheels) consisting of superparamagnetic particles assembled and powered by rotating ac magnetic fields, is to employ a nearby surface to provide friction. Here, we used total internal reflection microscopy to show that individual 8.3 μm particles roll inefficiently with significant slip because of a particle-surface fluid gap of 20-80 nm. We determined that both gap width and slip increase with the increasing particle rotation rate when the load force is provided by gravity alone, thus providing an upper bound on translational velocity. By imposing an additional load force with a dc magnetic field gradient superimposed on the ac field, we were able to decrease the gap width and thereby enhance translation velocities. For example, an additional load force of 0.2 F provided by a dc field gradient increased the translational velocity from 40 to 80 μm/s for a 40 Hz rotation rate. The translation velocity increases with the decreasing gap width whether the gap is varied by dc field gradient-induced load forces or by reducing the Debye length with salt. These results present a strategy to accelerate surface-enabled rolling of microscale particles and open the possibility of high-speed μwheel rolling independent of the gravitational field.
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http://dx.doi.org/10.1021/acs.langmuir.8b04084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536127PMC
March 2019

A local and global sensitivity analysis of a mathematical model of coagulation and platelet deposition under flow.

PLoS One 2018 26;13(7):e0200917. Epub 2018 Jul 26.

Department of Applied Mathematics and Statistics, Colorado School of Mines, Golden, CO, United States of America.

The hemostatic response involves blood coagulation and platelet aggregation to stop blood loss from an injured blood vessel. The complexity of these processes make it difficult to intuit the overall hemostatic response without quantitative methods. Mathematical models aim to address this challenge but are often accompanied by numerous parameters choices and thus need to be analyzed for sensitivity to such choices. Here we use local and global sensitivity analyses to study a model of coagulation and platelet deposition under flow. To relate with clinical assays, we measured the sensitivity of three specific thrombin metrics: lag time, maximum relative rate of generation, and final concentration after 20 minutes. In addition, we varied parameters of three different classes: plasma protein levels, kinetic rate constants, and platelet characteristics. In terms of an overall ranking of the model's sensitivities, we found that the local and global methods provided similar information. Our local analysis, in agreement with previous findings, shows that varying parameters within 50-150% of baseline values, in a one-at-a-time (OAT) fashion, always leads to significant thrombin generation in 20 minutes. Our global analysis gave a different and novel result highlighting groups of parameters, still varying within the normal 50-150%, that produced little or no thrombin in 20 minutes. Variations in either plasma levels or platelet characteristics, using either OAT or simultaneous variations, always led to strong thrombin production and overall, relatively low output variance. Simultaneous variation in kinetics rate constants or in a subset of all three parameter classes led to the highest overall output variance, incorporating instances with little to no thrombin production. The global analysis revealed multiple parameter interactions in the lag time and final concentration leading to relatively high variance; high variance was also observed in the thrombin generation rate, but parameters attributed to that variance acted independently and additively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200917PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062055PMC
January 2019

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model.

Arterioscler Thromb Vasc Biol 2018 05 22;38(5):1052-1062. Epub 2018 Feb 22.

From the Chemical and Biological Engineering Department, Colorado School of Mines, Golden (M.L., R.M.S., P.J.K., A.M.W., J.R.S., K.B.N.)

Objective: The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis.

Approach And Results: A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor-coated valve sinus. Platelets accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer.

Conclusions: A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.
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http://dx.doi.org/10.1161/ATVBAHA.118.310731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5920765PMC
May 2018

Enhanced Fibrinolysis with Magnetically Powered Colloidal Microwheels.

Small 2017 09 18;13(36). Epub 2017 Jul 18.

Chemical and Biological Engineering Department, Colorado School of Mines, 1500 Illinois St., Golden, CO, 80401, USA.

Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (µwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 µg mL tPA. µWheels following corkscrew trajectories can also penetrate through 100 µm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal.
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http://dx.doi.org/10.1002/smll.201700954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927958PMC
September 2017

Flow chamber and microfluidic approaches for measuring thrombus formation in genetic bleeding disorders.

Platelets 2017 Jul 22;28(5):463-471. Epub 2017 May 22.

a Chemical and Biological Engineering Department , Colorado School of Mines , Golden , CO , USA.

Platelet adhesion and aggregation, coagulation, fibrin formation, and fibrinolysis are regulated by the forces and flows imposed by blood at the site of a vascular injury. Flow chambers designed to observe these events are an indispensable part of doing hemostasis and thrombosis research, especially with human blood. Microfluidic methods have provided the flexibility to design flow chambers with complex geometries and features that more closely mimic the anatomy and physiology of blood vessels. Additionally, microfluidic systems with integrated optics and/or pressure sensors and on-board signal processing could transform what have been primarily research tools into clinical assays. Here, we describe a historical review of how flow-based approaches have informed biophysical mechanisms in genetic bleeding disorders, challenges and potential solutions for developing models of bleeding in vitro, and outstanding issues that need to be addressed prior to their use in clinical settings.
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http://dx.doi.org/10.1080/09537104.2017.1306042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6131111PMC
July 2017

Magnetic Microlassos for Reversible Cargo Capture, Transport, and Release.

Langmuir 2017 06 27;33(23):5932-5937. Epub 2017 Mar 27.

Department of Chemical and Biological Engineering, Colorado School of Mines , Golden, Colorado 80401, United States.

Microbot propulsion has seen increasing interest in recent years as artificial methods that overcome the well-established reversible and challenging nature of microscale fluid mechanics. While controlled movement is an important feature of microbot action, many envisioned applications also involve cargo transport where microbots must be able to load and unload contents on command while tolerating complex solution chemistry. Here we introduce a physical method that uses flexible and linked superparamagnetic colloidal chains, which can form closed rings or "lassos" in the presence of a planar rotating magnetic field. By adding an additional AC magnetic field along the direction perpendicular to the substrate, we can orient the lasso at a tilted camber angle. We show that these magnetic lassos can roll at substantial velocities, with precise spatial control by manipulating both field strength and phase lag. Moreover, the lasso can curl around and capture cargo tightly and transport it based on a wheel-type mechanism. At the targeted destination, cargo is easily released upon field removal and the lasso can be readily reused. Since the entire process is physically controlled with no chemistry for attachment or disengagement involved, our system can potentially be used for transporting diverse types of cargo under different solution conditions.
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http://dx.doi.org/10.1021/acs.langmuir.7b00357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931268PMC
June 2017

Elevated hematocrit enhances platelet accumulation following vascular injury.

Blood 2017 05 1;129(18):2537-2546. Epub 2017 Mar 1.

Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro and in silico. Compared with controls, mice with elevated hematocrit (RBC) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBC mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans.
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http://dx.doi.org/10.1182/blood-2016-10-746479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418635PMC
May 2017

Ask1 regulates murine platelet granule secretion, thromboxane A generation, and thrombus formation.

Blood 2017 03 27;129(9):1197-1209. Epub 2016 Dec 27.

Cardeza Center for Vascular Biology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA.

Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of ( ) resulted in a marked functional defect in platelets. platelets showed an impaired agonist-induced integrin αβ activation and platelet aggregation. Although there was no difference in Ca rise, platelet granule secretion and thromboxane A (TxA) generation were significantly attenuated in platelets. The defective granule secretion observed in platelets was a consequence of impaired TxA generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in platelets. On the contrary, activation of c-Jun -terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA in platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl-induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis.
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http://dx.doi.org/10.1182/blood-2016-07-729780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374734PMC
March 2017

Non reciprocal skewed rolling of a colloidal wheel due to induced chirality.

Soft Matter 2016 Nov;12(46):9314-9320

Institute of Physics Universität Bayreuth, 95440 Bayreuth, Germany.

We use a rotating magnetic field to assemble an oblate cluster of paramagnetic colloidal particles. If the field is rotating about a horizontal axis, the cluster acts as a colloidal wheel rolling across the supporting glass surface. The motion is reversible upon switching the direction of rotation. Surprisingly, the reversibility is lost if the axis of field rotation is tilted with respect to the surface. The wheel then rolls in a direction that is not perpendicular to the field rotation axis. We explain the skewed rotation with an interplay between a magnetic driving torque, magnetic anisotropy and an anisotropy in the hydrodynamic mobility tensor in the vicinity of a surface. The opposing forward and backward drive induce opposite chirality in the degrees of freedom of the mechanically achiral colloidal wheel.
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http://dx.doi.org/10.1039/c6sm02143cDOI Listing
November 2016

Bead-Based Microfluidic Sediment Analogues: Fabrication and Colloid Transport.

Langmuir 2016 09 1;32(36):9342-50. Epub 2016 Sep 1.

Department of Chemical and Biological Engineering, Colorado School of Mines , Golden, Colorado 80401, United States.

Mobile colloids can act as carriers for low-solubility contaminants in the environment. However, the dominant mechanism for this colloid-facilitated transport of chemicals is unclear. Therefore, we developed a bead-based microfluidic platform of sediment analogues and measured both single and population transport of model colloids. The porous medium is assembled through a bead-by-bead injection method. This approach has the versatility to build both electrostatically homogeneous and heterogeneous media at the pore scale. A T-junction at the exit also allowed for encapsulation and enumeration of colloids effluent at single particle resolution to give population dynamics. Tortuosity calculated from pore-scale trajectory analysis and its comparison with lattice Boltzmann simulations revealed that transport of colloids was influenced by the size exclusion effect. The porous media packed by positively and negatively charged beads into two layers showed distinctive colloidal particle retention and significant remobilization and re-adsorption of particles during water flushing. We demonstrated the potential of our method to fabricate porous media with surface heterogeneities at the pore scale. With both single and population dynamics measurement, our platform has the potential to connect pore-scale and macroscale colloid transport on a lab scale and to quantify the impact of grain surface heterogeneities that are natural in the subsurface environment.
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http://dx.doi.org/10.1021/acs.langmuir.6b02184DOI Listing
September 2016

Blood flow and mass transfer regulation of coagulation.

Blood Rev 2016 09 29;30(5):357-68. Epub 2016 Apr 29.

Chemical and Biological Engineering, Colorado School of Mines, Golden, CO, USA; Pediatrics, University of Colorado-Denver, Aurora, CO, USA. Electronic address:

Blood flow regulates coagulation and fibrin formation by controlling the transport, or mass transfer, of zymogens, co-factors, enzymes, and inhibitors to, from, and within a growing thrombus. The rate of mass transfer of these solutes relative to their consumption or production by coagulation reactions determines, in part, the rate of thrombin generation, fibrin deposition, and thrombi growth. Experimental studies on the influence of blood flow on specific coagulation reactions are reviewed here, along with a theoretical framework that predicts how flow influences surface-bound coagulation binding and enzymatic reactions. These flow-mediated transport mechanisms are also used to interpret the role of binding site densities and injury size on initiating coagulation and fibrin deposition. The importance of transport of coagulation proteins within the interstitial spaces of thrombi is shown to influence thrombi architecture, growth, and arrest.
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http://dx.doi.org/10.1016/j.blre.2016.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023459PMC
September 2016

On-chip recalcification of citrated whole blood using a microfluidic herringbone mixer.

Biomicrofluidics 2015 Nov 18;9(6):064106. Epub 2015 Nov 18.

Chemical and Biological Engineering Department, Colorado School of Mines , Golden, Colorado 80401, USA.

In vitro assays of platelet function and coagulation are typically performed in the presence of an anticoagulant. The divalent cation chelator sodium citrate is among the most common because its effect on coagulation is reversible upon reintroduction of divalent cations. Adding divalent cations into citrated blood by batch mixing leads to platelet activation and initiation of coagulation after several minutes, thus limiting the time blood can be used before spontaneously clotting. In this work, we describe a herringbone microfluidic mixer to continuously introduce divalent cations into citrated blood. The mixing ratio, defined as the ratio of the volumetric flow rates of citrated blood and recalcification buffer, can be adjusted by changing the relative inlet pressures of these two solutions. This feature is useful in whole blood assays in order to account for differences in hematocrit, and thus viscosity. The recalcification process in the herringbone mixer does not activate platelets. The advantage of this continuous mixing approach is demonstrated in microfluidic vascular injury model in which platelets and fibrin accumulate on a collagen-tissue factor surface under flow. Continuous recalcification with the herringbone mixer allowed for flow assay times of up to 30 min, more than three times longer than the time achieved by batch recalcification. This continuous mixer allows for measurements of thrombus formation, remodeling, and fibrinolysis in vitro over time scales that are relevant to these physiological processes.
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http://dx.doi.org/10.1063/1.4935863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4654733PMC
November 2015

In microfluidico: Recreating in vivo hemodynamics using miniaturized devices.

Biorheology 2015 ;52(5-6):303-18

Institute for Medicine and Engineering, Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA, USA.

Microfluidic devices create precisely controlled reactive blood flows and typically involve: (i) validated anticoagulation/pharmacology protocols, (ii) defined reactive surfaces, (iii) defined flow-transport regimes, and (iv) optical imaging. An 8-channel device can be run at constant flow rate or constant pressure drop for blood perfusion over a patterned collagen, collagen/kaolin, or collagen/tissue factor (TF) to measure platelet, thrombin, and fibrin dynamics during clot growth. A membrane-flow device delivers a constant flux of platelet agonists or coagulation enzymes into flowing blood. A trifurcated device sheaths a central blood flow on both sides with buffer, an ideal approach for on-chip recalcification of citrated blood or drug delivery. A side-view device allows clotting on a porous collagen/TF plug at constant pressure differential across the developing clot. The core-shell architecture of clots made in mouse models can be replicated in this device using human blood. For pathological flows, a stenosis device achieves shear rates of >100,000 s(-1) to drive plasma von Willebrand factor (VWF) to form thick long fibers on collagen. Similarly, a micropost-impingement device creates extreme elongational and shear flows for VWF fiber formation without collagen. Overall, microfluidics are ideal for studies of clotting, bleeding, fibrin polymerization/fibrinolysis, cell/clot mechanics, adhesion, mechanobiology, and reaction-transport dynamics.
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http://dx.doi.org/10.3233/BIR-15065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814229PMC
November 2016

High-throughput linear optical stretcher for mechanical characterization of blood cells.

Cytometry A 2016 04 13;89(4):391-7. Epub 2015 Nov 13.

Chemical and Biological Engineering Department, Colorado School of Mines, Golden, Colorado, 80401.

This study describes a linear optical stretcher as a high-throughput mechanical property cytometer. Custom, inexpensive, and scalable optics image a linear diode bar source into a microfluidic channel, where cells are hydrodynamically focused into the optical stretcher. Upon entering the stretching region, antipodal optical forces generated by the refraction of tightly focused laser light at the cell membrane deform each cell in flow. Each cell relaxes as it flows out of the trap and is compared to the stretched state to determine deformation. The deformation response of untreated red blood cells and neutrophils were compared to chemically treated cells. Statistically significant differences were observed between normal, diamide-treated, and glutaraldehyde-treated red blood cells, as well as between normal and cytochalasin D-treated neutrophils. Based on the behavior of the pure, untreated populations of red cells and neutrophils, a mixed population of these cells was tested and the discrete populations were identified by deformability. © 2015 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.22794DOI Listing
April 2016

Physiochemical artifacts in FeCl3 thrombosis models.

Authors:
Keith B Neeves

Blood 2015 Aug;126(6):700-1

COLORADO SCHOOL OF MINES.

In this issue of Blood, Ciciliano et al demonstrate that thrombus formation in ferric chloride (FeCl3) thrombosis models relies on physiochemical, rather than biological, mechanisms.
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http://dx.doi.org/10.1182/blood-2015-05-644708DOI Listing
August 2015

Fluid Mechanics of Blood Clot Formation.

Annu Rev Fluid Mech 2015 Jan;47:377-403

Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado 80401.

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.
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http://dx.doi.org/10.1146/annurev-fluid-010814-014513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519838PMC
January 2015

Microfluidic technology as an emerging clinical tool to evaluate thrombosis and hemostasis.

Thromb Res 2015 Jul 21;136(1):13-9. Epub 2015 May 21.

Dept. of Pediatrics - Hematology/Oncology, University of Colorado School of Medicine, Aurora, CO, USA; University of Colorado Hemophilia and Thrombosis Center, Aurora, CO, USA; Graduate Program- Human Medical Genetics and Genomics, University of Colorado School of Medicine, Aurora, CO, USA. Electronic address:

Assessment of platelet function and coagulation under flow conditions can augment traditional static assays used to evaluate patients with suspected hemostatic or thrombotic disorders. Among the available flow-based assays, microfluidic devices require the smallest blood volume and provide multiple output options. These assays are based on the presence of wall shear stress that mimics in vivo interactions between blood components and vessel walls. Microfluidic devices can generate essential information regarding homeostatic regulation of platelet activation and subsequent engagement of the coagulation cascade leading to fibrin deposition and clot formation. Emerging data suggest that microfluidic assays may also reveal consistent patterns of hemostatic or thrombotic pathology, and could aid in assessing and monitoring patient-specific effects of coagulation-modifying therapies.
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http://dx.doi.org/10.1016/j.thromres.2015.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910695PMC
July 2015

Platelet bioreactor-on-a-chip.

Blood 2014 Sep;124(12):1857-67

Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition,micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4168343PMC
http://dx.doi.org/10.1182/blood-2014-05-574913DOI Listing
September 2014

Elastic behavior and platelet retraction in low- and high-density fibrin gels.

Biophys J 2015 Jan;108(1):173-83

Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, Colorado; Department of Pediatrics, University of Colorado, Aurora, Colorado. Electronic address:

Fibrin is a biopolymer that gives thrombi the mechanical strength to withstand the forces imparted on them by blood flow. Importantly, fibrin is highly extensible, but strain hardens at low deformation rates. The density of fibrin in clots, especially arterial clots, is higher than that in gels made at plasma concentrations of fibrinogen (3-10 mg/mL), where most rheology studies have been conducted. Our objective in this study was to measure and characterize the elastic regimes of low (3-10 mg/mL) and high (30-100 mg/mL) density fibrin gels using shear and extensional rheology. Confocal microscopy of the gels shows that fiber density increases with fibrinogen concentration. At low strains, fibrin gels act as thermal networks independent of fibrinogen concentration. Within the low-strain regime, one can predict the mesh size of fibrin gels by the elastic modulus using semiflexible polymer theory. Significantly, this provides a link between gel mechanics and interstitial fluid flow. At moderate strains, we find that low-density fibrin gels act as nonaffine mechanical networks and transition to affine mechanical networks with increasing strains within the moderate regime, whereas high-density fibrin gels only act as affine mechanical networks. At high strains, the backbone of individual fibrin fibers stretches for all fibrin gels. Platelets can retract low-density gels by >80% of their initial volumes, but retraction is attenuated in high-density fibrin gels and with decreasing platelet density. Taken together, these results show that the nature of fibrin deformation is a strong function of fibrin fiber density, which has ramifications for the growth, embolization, and lysis of thrombi.
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http://dx.doi.org/10.1016/j.bpj.2014.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286595PMC
January 2015