Concizumab improves clot formation in hemophilia A under flow.

BACKGROUND: Inhibition of tissue factor pathway inhibitor (TFPI) is an emerging therapeutic strategy for treatment of hemophilia. Concizumab is a monoclonal antibody that binds TFPI and blocks its inhibition of factor (F)Xa thereby extending the initiation of coagulation and compensating for lack of FVIII or FIX. OBJECTIVES: The objective of this in vitro study was to evaluate how concizumab affects clot formation in hemophilia A under flow. METHODS: Blood was collected from normal controls or people with hemophilia A. An anti-FVIII antibody was added to normal controls to simulate hemophilia A with inhibitory antibodies to FVIII. Whole blood added activated recombinant factor VII (rFVIIa, 25 nM) or concizumab (200, 1000, 4000 ng/mL) were perfused at 100 s-1 over a surface micropatterned with tissue factor (TF) and collagen related peptide. Platelet and fibrin(ogen) accumulation were measured by confocal microscopy. Static thrombin generation in plasma was measured in response to rFVIIa and concizumab. RESULTS: Concizumab (1000, 4000 ng/mL) and rFVIIa both rescued (93-101%) total platelet accumulation, but only partially rescued (53-63%) fibrin(ogen) incorporation to normal control levels in simulated hemophilia A. Results in congenital haemophilia A blood confirmed effects of rFVIIa and concizumab. While these two agents had similar effect on clot formation under flow, concizumab enhanced thrombin generation in plasma under static conditions to a greater extent than rFVIIa. CONCLUSIONS: TFPI inhibition by concizumab enhanced activation and aggregation of platelets and fibrin clot formation in hemophilia A to levels comparable to that of rFVIIa.

Magnetically powered microwheel thrombolysis of occlusive thrombi in zebrafish.

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here, we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-functionalized µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-functionalized µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Reversible Microwheel Translation Induced by Polymer Depletion.

For in vivo applications, microbots (µbots) must move, which is a need that has led to designs, such as helical swimmers, that translate through the bulk fluid. We have previously demonstrated that, upon application of a rotating magnetic field, colloidal particles in aqueous systems can be reversibly assembled from superparamagnetic particles into µbots that translate along surfaces using wet friction. Here, we show that high-molecular-weight polymers of a size that approaches the length scale of the gap between the µbot and surface can be excluded, impacting µbot transport. Using xanthan gum as a convenient high-molecular-weight model, we determine that polymer depletion imparts only a weak effect on colloid-surface interactions but has a significant influence on local viscosity, which is an effect great enough to induce a reversal in the µbot translation direction.

Magnetically Powered Microwheel Thrombolysis of Occlusive Thrombi in Zebrafish.

Tissue plasminogen activator (tPA) is the only FDA approved treatment for ischemic stroke but carries significant risks, including major hemorrhage. Additional options are needed, especially in small vessel thrombi which account for ~25% of ischemic strokes. We have previously shown that tPA-functionalized colloidal microparticles can be assembled into microwheels (µwheels) and manipulated under the control of applied magnetic fields to enable rapid thrombolysis of fibrin gels in microfluidic models of thrombosis. Providing a living microfluidic analog, transparent zebrafish larvae have a highly conserved coagulation cascade that enables studies of hemostasis and thrombosis in the context of intact vasculature, clotting factors, and blood cells. Here we show that tPA-functionalized µwheels can perform rapid and targeted recanalization in vivo. This effect requires both tPA and µwheels, as minimal to no recanalization is achieved with tPA alone, µwheels alone, or tPA-functionalized microparticles in the absence of a magnetic field. We evaluated tPA-µwheels in CRISPR-generated plasminogen (plg) heterozygous and homozygous mutants and confirmed that tPA-µwheels are dose-dependent on plasminogen for lysis. We have found that magnetically powered µwheels as a targeted tPA delivery system are dramatically more efficient at plasmin-mediated thrombolysis than systemic delivery in vivo. Further development of this system in fish and mammalian models could enable a less invasive strategy for alleviating ischemia that is safer than directed thrombectomy or systemic infusion of tPA.

Magnetically Powered Chitosan Milliwheels for Rapid Translation, Barrier Function Rescue, and Delivery of Therapeutic Proteins to the Inflamed Gut Epithelium.

Inflammatory bowel disease (IBD) is mediated by an overexpression of tumor necrosis factor-α (TNF) by mononuclear cells in the intestinal mucosa. Intravenous delivery of neutralizing anti-TNF antibodies can cause systemic immunosuppression, and up to one-third of people are non-responsive to treatment. Oral delivery of anti-TNF could reduce adverse effects; however, it is hampered by antibody degradation in the harsh gut environment during transit and poor bioavailability. To overcome these shortcomings, we demonstrate magnetically powered hydrogel particles that roll along mucosal surfaces, provide protection from degradation, and sustain the local release of anti-TNF. Iron oxide particles are embedded into a cross-linked chitosan hydrogel and sieved to produce 100-200 µm particles called milliwheels (m-wheels). Once loaded with anti-TNF, these m-wheels release 10 to 80% of their payload over 1 week at a rate that depends on the cross-linking density and pH. A rotating magnetic field induces a torque on the m-wheels that results in rolling velocities greater than 500 µm/s on glass and mucus-secreting cells. The permeability of the TNF-challenged gut epithelial cell monolayers was rescued in the presence of anti-TNF carrying m-wheels, which both neutralized the TNF and created an impermeable patch over leaky cell junctions. With the ability to translate over mucosal surfaces at high speed, provide sustained release directly to the inflamed epithelium, and provide barrier rescue, m-wheels demonstrate a potential strategy to deliver therapeutic proteins for the treatment of IBD.

Zoster-Associated Prothrombotic Plasma Exosomes and Increased Stroke Risk.

Herpes zoster (HZ; shingles) caused by varicella zoster virus reactivation increases stroke risk for up to 1 year after HZ. The underlying mechanisms are unclear, however, the development of stroke distant from the site of zoster (eg, thoracic, lumbar, sacral) that can occur months after resolution of rash points to a long-lasting, virus-induced soluble factor (or factors) that can trigger thrombosis and/or vasculitis. Herein, we investigated the content and contributions of circulating plasma exosomes from HZ and non-HZ patient samples. Compared with non-HZ exosomes, HZ exosomes (1) contained proteins conferring a prothrombotic state to recipient cells and (2) activated platelets leading to the formation of platelet-leukocyte aggregates. Exosomes 3 months after HZ yielded similar results and also triggered cerebrovascular cells to secrete the proinflammatory cytokines, interleukin 6 and 8. These results can potentially change clinical practice through addition of antiplatelet agents for HZ and initiatives to increase HZ vaccine uptake to decrease stroke risk.

Coupling magnetic torque and force for colloidal microbot assembly and manipulation.

For targeted transport in the body, biomedical microbots (µbots) must move effectively in three-dimensional (3D) microenvironments. Swimming µbots translate via asymmetric or screw-like motions while rolling ones use friction with available surfaces to generate propulsive forces. We have previously shown that planar rotating magnetic fields assemble µm-scale superparamagnetic beads into circular µbots that roll along surfaces. In this, gravity is required to pull µbots near the surface; however, this is not necessarily practical in complex geometries. Here we show that rotating magnetic fields, in tandem with directional magnetic gradient forces, can be used to roll µbots on surfaces regardless of orientation. Simplifying implementation, we use a spinning permanent magnet to generate differing ratios of rotating and gradient fields, optimizing control for different environments. This use of a single magnetic actuator sidesteps the need for complex electromagnet or tandem field setups, removes requisite gravitational load forces, and enables µbot targeting in complex 3D biomimetic microenvironments.

Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α1-antitrypsin-derived peptide motif.

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α1-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Apolipoprotein A-I, elevated in trauma patients, inhibits platelet activation and decreases clot strength.

Apolipoprotein A-I (ApoA-I) is elevated in the plasma of a subgroup of trauma patients with systemic hyperfibrinolysis. We hypothesize that apoA-I inhibits platelet activation and clot formation. The effects of apoA-I on human platelet activation and clot formation were assessed by whole blood thrombelastography (TEG), platelet aggregometry, P-selectin surface expression, microfluidic adhesion, and Akt phosphorylation. Mouse models of carotid artery thrombosis and pulmonary embolism were used to assess the effects of apoA-I in vivo. The ApoA-1 receptor was investigated with transgenic mice knockouts (KO) for the scavenger receptor class B member 1 (SR-BI). Compared to controls, exogenous human apoA-I inhibited arachidonic acid and collagen-mediated human and mouse platelet aggregation, decreased P-selectin surface expression and Akt activation, resulting in diminished clot strength and increased clot lysis by TEG. ApoA-I also decreased platelet aggregate size formed on a collagen surface under flow. In vivo, apoA-I delayed vessel occlusion in an arterial thrombosis model and conferred a survival advantage in a pulmonary embolism model. SR-BI KO mice significantly reduced apoA-I inhibition of platelet aggregation versus wild-type platelets. Exogenous human apoA-I inhibits platelet activation, decreases clot strength and stability, and protects mice from arterial and venous thrombosis via the SR-BI receptor.

Breaking the fibrinolytic speed limit with microwheel co-delivery of tissue plasminogen activator and plasminogen.

BACKGROUND: To reestablish blood flow in vessels occluded by clots, tissue plasminogen activator (tPA) can be used; however, its efficacy is limited by transport to and into a clot and by the depletion of its substrate, plasminogen. OBJECTIVES: To overcome these rate limitations, a platform was designed to co-deliver tPA and plasminogen based on microwheels (µwheels), wheel-like assemblies of superparamagnetic colloidal beads that roll along surfaces at high speeds. METHODS: The biochemical speed limit was determined by measuring fibrinolysis of plasma clots at varying concentrations of tPA (10-800 nM) and plasminogen (1-6 µM). Biotinylated magnetic mesoporous silica nanoparticles were synthesized and bound to streptavidin-coated superparamagnetic beads to make studded beads. Studded beads were loaded with plasminogen and tPA was immobilized on their surface. Plasminogen release and tPA activity were measured on the studded beads. Studded beads were assembled into µwheels with rotating magnetic fields and fibrinolysis of plasma clots was measured in a microfluidic device. RESULTS: The biochemical speed limit for plasma clots was ~15 µm/min. Plasminogen-loaded, tPA-immobilized µwheels lyse plasma clots at rates comparableto the biochemical speed limit. With the addition of a corkscrew motion, µwheels penetrate clots, thereby exceeding the biochemical speed limit (~20 µm/min) and achieving lysis rates 40-fold higher than 50 nM tPA. CONCLUSIONS: Co-delivery of an immobilized enzyme and its substrate via a microbot capable of mechanical work has the potential to target and rapidly lyse clots that are inaccessible by mechanical thrombectomy devices or recalcitrant to systemic tPA delivery.

Isotopically nonstationary 13C metabolic flux analysis in resting and activated human platelets.

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.

Delivery and actuation of aerosolized microbots.

For disease of the lung, the physical key to effective inhalation-based therapy is size; too large (10's of µm) and the particles or droplets do not remain suspended in air to reach deep within the lungs, too small (subµm) and they are simply exhaled without deposition. µBots within this ideal low-µm size range however are challenging to fabricate and would lead to devices that lack the speed and power necessary for performing work throughout the pulmonary network. To uncouple size from structure and function, here we demonstrate an approach where individual building blocks are aerosolized and subsequently assembled in situ into µbots capable of translation, drug delivery, and mechanical work deep within lung mimics. With this strategy, a variety of pulmonary diseases previously difficult to treat may now be receptive to µbot-based therapies.

An "occlusive thrombosis-on-a-chip" microfluidic device for investigating the effect of anti-thrombotic drugs.

Cardiovascular disease remains one of the world's leading causes of death. Myocardial infarction (heart attack) is triggered by occlusion of coronary arteries by platelet-rich thrombi (clots). The development of new anti-platelet drugs to prevent myocardial infarction continues to be an active area of research and is dependent on accurately modelling the process of clot formation. Occlusive thrombi can be generated in vivo in a range of species, but these models are limited by variability and lack of relevance to human disease. Although in vitro models using human blood can overcome species-specific differences and improve translatability, many models do not generate occlusive thrombi. In those models that do achieve occlusion, time to occlusion is difficult to measure in an unbiased and objective manner. In this study we developed a simple and robust approach to determine occlusion time of a novel in vitro microfluidic assay. This highlighted the potential for occlusion to occur in thrombosis microfluidic devices through off-site coagulation, obscuring the effect of anti-platelet drugs. We therefore designed a novel occlusive thrombosis-on-a-chip microfluidic device that reliably generates occlusive thrombi at arterial shear rates by quenching downstream coagulation. We further validated our device and methods by using the approved anti-platelet drug, eptifibatide, recording a significant difference in the "time to occlude" in treated devices compared to control conditions. These results demonstrate that this device can be used to monitor the effect of antithrombotic drugs on time to occlude, and, for the first time, delivers this essential data in an unbiased and objective manner.

In vitro flow-based assay: From simple toward more sophisticated models for mimicking hemostasis and thrombosis.

In vitro flow-based assays are widely used to investigate the role of platelets and coagulation in hemostasis and thrombosis. Their main advantage over other assays relies on the fact that they integrate blood flow that regulates many aspects of platelet function, including adhesion, activation, and aggregation. Blood flow is also central in the regulation of coagulation through its ability to modulate the local concentrations of coagulation factors within and around thrombi. The most broadly used assay to study thrombus formation consists in perfusing whole blood over immobilized fibrillar collagen through a single channel, which helps to reproduce thrombus formation as it occurs in vivo after vascular injury, with platelets adhering, becoming activated, and forming a mural thrombus. This process can also be studied under conditions of thrombin generation, notably by recalcifying blood collected in sodium citrate. In this manuscript, we briefly discuss the advantages and limits of this broadly used "in vitro thrombus formation model." The main emphasis is on the description of the most recent developments regarding design of new flow models and new techniques, and how these may advance the landscape of in vitro studies into the formation of physiological or pathophysiological thrombi. Challenges linked to mimicking the formation of a hemostatic plug in a healthy vessel or a thrombus in diseased arteries and the complexity of reproducing the various aspects of venous thrombosis are discussed. Future directions are proposed to improve the physiological or pathophysiological relevance of current flow-based assays.

Physical forces regulating hemostasis and thrombosis: Vessels, cells, and molecules in illustrated review.

This illustrated review focuses on the physical forces that regulate hemostasis and thrombosis. These phenomena span from the vessel to the cellular to the molecular scales. Blood is a complex fluid with a viscosity that varies with how fast it flows and the size of the vessel through which it flows. Blood flow imposes forces on the vessel wall and blood cells that dictates the kinetics, structure, and stability of thrombi. The mechanical properties of blood cells create a segmented flowing fluid whereby red blood cells concentrate in the vessel core and platelets marginate to the near-wall region. At the vessel wall, shear stresses are highest, which requires a repertoire of receptors with different bond kinetics to roll, tether, adhere, and activate on inflamed endothelium and extracellular matrices. As a thrombus grows and then contracts, forces regulate platelet aggregation as well as von Willebrand factor function and fibrin mechanics. Forces can also originate from platelets as they respond to the external forces and sense the stiffness of their local environment.

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

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.

Computationally Driven Discovery in Coagulation.

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).

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

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 (θmax = 0.051) was measured directly. The surface-blocking function and the deposition coefficient (kpore = 3.56 s-1) 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 via coupling of direct pore-scale measurements and an advection-dispersion-deposition model.