Keith B. Neeves - US grants

[unreadable] DESCRIPTION (provided by applicant): DESCRIPTION. The goal of the proposed research is to understand how the generation of thrombin at the surface of platelets stabilizes blood clots. The primary roles of thrombin in clot formation are to catalyze fibrin polymerization and activate platelets. Which of these two mechanisms is most responsible for clot stability and under what flow conditions is unknown. The central hypothesis for the proposed research is that at venous shear stresses, stable clots can form in the absence of platelet activation but at arterial shear stress, platelet activation is the predominant mechanism for stabilizing clots. To study clot stability, we will use a novel microfluidic tool to introduce soluble factors into flowing blood. The rationale for this approach is that blood is a moving biological fluid in vivo, and therefore it is important to study blood phenotype in vitro under physiologically relevant flow conditions. The formation and morphology of the resultant clots will be measured by phase contrast, fluorescence, and electron microscopy methods. The stability of clot will be determined by analyzing its fragmentation under high shear stress. This innovative approach is expected to yield the following outcomes. In specific aim 1, we will determine how thrombin flux.affects fibrin polymerization, which is important because it will identify the amount of thrombin generation needed under flow to form a fibrin clot. In specific aim 2, we will decouple fibrin polymerization from platelet activation to determine the relative role of these two mechanisms in forming stable clots. By isolating the mechanism(s) that are responsible for clot stability at different flow conditions we expect to gain further insight into the pathology of bleeding disorders and embolism. LAY SUMMARY. The goal of this research is to understand the mechanisms responsible for forming stable blood clots. Poor clot stability is the result of bleeding disorders like hemophilia and the cause of stroke by emboli shedding from preexisting clots. By unraveling the mechanisms responsible for clot stability, this research will identify new therapeutic targets for individuals who suffer from these diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Ischemic strokes can be treated with either chemical or mechanical means, each with advantages and disadvantages. Tissue plasminogen activator (tPA), a common clot buster, has been used to treat thrombotic clots but can lead to excessive bleeding and must be used soon after symptoms first occur. Mechanical methods can restore blood flow quickly but are invasive and can leave residual prothrombotic material on vessel walls, increasing risk for secondary stroke. To address these drawbacks, we propose a targeted delivery approach performed through an injectable colloidal solution controlled by an external magnetic field. This non-invasive approach combines pharmacological and mechanical methods for clot removal. Here, individual particles in solution are injected into the blood and, upon application of a magnetic field, self-assemble into small microdevices capable of targeting fibrinolytic agents and mechanically attacking a clot in the absence of catheters. As both microdevice assembly and driving forces are provided by the external field, once the procedure is finished, devices self- disassemble into small building blocks removable by the body via phagocytosis. We note that, as the approach is microscale in nature, it can be tuned to more carefully remove any prothrombotic residual clot that can arise in mechanical thrombectomies. Our aims include: Specific Aim 1: Determine the rate at which colloidal-based devices mechanically remove clots. We will investigate clot removal rate by mechanical disruption as a function of operating parameters such as microdevice size and spin-rate within microfluidic vascular mimics. Specific Aim 2: Determine the effectiveness with which fibrinolytic-modified colloidal microdevices can be used to enhance clot removal. Here, we will synthesize tPA-modified magnetic beads and demonstrate their use as fibrinolytic agents within microfluidic vascular mimics. We expect direct coupling of tPA to enhance dissolution rates over mechanical disruption alone. Aim 3: Demonstrate device assembly and targeting within in vivo environments. With a well-established animal stroke model we will demonstrate the delivery, assembly, and targeting of magnetic assemblies to the site of vascular occlusion. Imaged with available small animal MRI facilities, these studies will provide the necessary proof-of-principle for further investigations.

DESCRIPTION (provided by applicant): In recent years, significant advances in understanding the molecular basis of bleeding disorders have been made, but a large portion of the variability in bleeding severity remains unexplained. In this project, the focus is on hemophilia and von Willebrand disease (VWD), where the observed variability in bleeding patterns cannot be assigned to a single measurable parameter. Clot formation is a complex, non-linear process seriously impaired in persons with these disorders. Because it involves the large biochemical pathway of coagulation coupled to platelet function and biophysical mechanisms including blood flow, it is well suited for study with a systems biology approach. The long-term goal of this research is to develop complementary computational and in vitro models that predict an individual's bleeding potential based on variables measured from their blood. The objective in this application is to identify biochemical and biophysical modifiers of bleeding in hemophilia and VWD. Potential modifiers include variables such as the composition of blood, platelet attributes, and the physical properties of clots. The central hypothesis is that our computational models that encompass the biochemical pathways of thrombus formation and platelet function coupled to the blood's fluid dynamics can identify the primary modifiers of bleeding patterns in these disorders. This hypothesis was formulated on the basis of preliminary data produced in the applicants' laboratories. The rationale for the proposed research is that the reductionist approach to predicting bleeding based on individual plasma components has failed. There is great detailed knowledge of the biochemical pathways that contribute to bleeding, but it is still not possible to reliably assign bleeding risk. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Develop and validate computational models of bleeding; 2) Identify modifiers of bleeding in hemophilia and VWD by computational sensitivity analyses; and 3) Predict clinical bleeding in a cohort of bleeding disorder patients. Under the first aim, existing models of thrombosis will be modified to simulate the unique biophysical environment of bleeding, defined by the transport of plasma proteins and blood cells into a porous extravascular space. Computational models will be validated against a microfluidic-based bleeding assay. Under the second aim, the computational models will be used to screen the large parameter space of variables known to affect clot formation. Parameters that significantly alter bleeding in the models will be tested experimentally, and, in the third aim, correlated to clinical bleeding patterns. The models will also be used to predict th response to therapy in a cohort of hemophilia patients with inhibitors. The approach is innovative because it represents a new and substantive departure from the status quo, namely a focus on the biophysical mechanisms of bleeding. The proposed research is significant because it is the first step in a continuum of research expected to lead ultimately to improved diagnosis and therapeutic strategies to prevent bleeding across a wide range of pathologies.

1351672
Neeves

Blood clots constitute an exquisitely engineered system, in which a complex fluid transforms into a solid plug at the site of an injury. Stable hemostatic clots are designed to arrest bleeding without occluding the vessel, withstand the forces of flowing blood, and slowly dissolve in concert with the wound healing process. Instabilities in any of these events can cause excessive clotting, or thrombosis, which is a leading cause of death. Despite the extensive knowledge base on the biochemistry and cell biology of clot formation, the mechanistic differences between a hemostatic clot and a thrombotic one remain largely unknown. Recent findings from the laboratory of the PI and from other labs suggest that impeding the transport of solutes away from the core of a clot is one mechanism that may prevent thrombosis. Based on this evidence, the hypothesis of the proposed studies is that transport of coagulation factors and platelet agonists within the interstitial space between blood cells is a key regulator of clot growth. If this hypothesis proves correct, then targeting this biophysical mechanism in conjunction with the conventional biochemical mechanisms could lead to more effective treatment of thrombosis.

Intellectual Merit:
This proposal investigates an important physiological system through the development of quantitative relationships between clot composition and growth. The conventional models of clot formation focus primarily on the kinetic processes involved in coagulation reactions and platelet signaling. The proposed studies build upon previous models by incorporating interstitial solute transport as a key mechanism of clot growth. With more capable predictive methods available, better drugs and drug delivery strategies can be developed. This hypothesis will be addressed by the following specific aims: (i) identifying the transport barriers that regulate clot growth and arrest, (ii) mapping the pore structure of clots, and (iii) exploiting interstitial transport to modulate clot growth. The implemented approach relies on applying theories and methods from the field of porous media transport to characterizing transport in tissues. In vitro and in vivo models of vascular injury will be used to measure transport properties in clots and the structure of their interstitial pore space. Constitutive relationships describing solute transport as a function of clot structure and composition will be developed for a range of physiological conditions. Results will be used to assess how known risk factors for thrombosis lead to uncontrolled clot growth and how this process can be physically impeded.

Broader Impacts:
The proposed studies will develop theoretical and experimental models to predict blood clot growth and test novel therapeutic strategies. This is a potentially transformative outcome since controlling thrombosis is one of the grand challenges in medicine. The research plan integrates with the education plan by creating K-12 outreach programs and undergraduate research opportunities focused on the interface between engineering and biology. Specific educational and outreach objectives include (i) improving middle school students' attitudes towards science with hands-on curriculum, (ii) developing and assessing inquiry-based learning program in bioengineering at a high school with predominantly Latino students, and (iii) establishing a summer undergraduate research program in cellular biomechanics in partnership with the Children's Hospital Colorado for students in the Multicultural Engineering Program at the Colorado School of Mines.

The PIs will create a self-sustaining scanning probe microscopy (SPM) facility with unique capabilities for advanced materials research at the Colorado School of Mines (CSM). This instrumentation request includes an atomic force microscope (AFM) coupled to an optical microscope for simultaneous imaging and physical properties measurements at the nanoscale for a wide range of advanced materials in renewable energy and life sciences. The proposed facility is designed for a multi-user environment and will be operated in a shared-use facility, with participation from the majority of departments across campus as well as our regional partners the National Renewable Energy Laboratory (NREL) and Children's Hospital Colorado (CHC). The PIs will organize and host an annual Rocky Mountain Scanning Probe Microscopy Workshop to provide training opportunities and attract new regional users to the facility beyond our partners at NREL and CHC. Undergraduates will be introduced to SPM through our existing summer REU programs.

The PIs will create a self-sustaining scanning probe microscopy (SPM) facility with unique capabilities for advanced materials research at the Colorado School of Mines (CSM). This instrumentation request includes an atomic force microscope (AFM) coupled to an optical microscope for simultaneous imaging and physical properties measurements at the nanoscale for a wide range of advanced materials. The unique feature of the proposed SPM facility is its ability to measure electrical and mechanical properties of materials in direct registry with topography at nanoscale resolution under their designed operating conditions (e.g illumination of photovoltaics and hydrated conditions for biological). The measurement of interfacial physical properties at small length scales is essential to the development of the next generation of materials for renewable energy and life sciences. This facility will transform the current empirical understanding of these complex materials into true science. Specific advanced capabilities beyond standard SPM modes (contact, tapping, phase) include optical microscopy, electro/mechanical property measurement, and environmental control. The microscope facilitates quick identification of the specific region of interest and enables optical stimulation to obtain properties such as a photoconductivity or fluorescence in registry with topology. Spatially resolved measurements of critical material properties such as conductivity, modulus, and viscoelasticity can be made as a function of temperature, humidity, and illumination intensity/wavelength.

Hormone therapies like oral contraception (OC) confer a heightened risk of venous thromboembolism (VTE) in premenopausal women. Until we gain mechanistic insights into why this happens, it is not possible to predict who is at risk for sex hormone-induced VTE. The long-term goal of this research is to identify the mechanisms by which sex hormones modulate hemostasis and thrombosis. The overall objective in this application is to determine how OC alter platelet function using a systems biology approach that combines ?omics technologies with computational models. Previous work on hormone-induced VTE have implicated several mechanisms related to platelet function including response to agonists, metabolism of arachidonic acid (AA), and gene expression. Systematic studies of these processes over different time scales and how they relate to each other is lacking and will be addressed here. The central hypothesis is that OC increase platelet reactivity over three time scales; (i) acutely (seconds-minutes) by potentiating calcium release from intracellular stores, (ii) metabolically (minutes-hours) by elevating thromboxane metabolism, and (iii) genomically (days-months) by altering the expression of adhesive receptors. This hypothesis is based on preliminary data that platelets incubated with physiologic concentrations of 17?-estradiol have higher intracellular calcium concentrations following adhesion to collagen and altered central metabolism. The rationale for the proposed research two- fold: (i) the development of new tools to study hormone-induced VTE over multiple time scales, and (ii) to measure the effects of exogenous hormones on platelet function over these time scales. This hypothesis will be tested by two specific aims: 1) Development of systems biology tools. 2) Measuring the effects of OC on platelet function over diverse time scales. Under the first aim, computational models of calcium dynamics and metabolism in platelets will be developed informed by experiments of platelet adhesion and metabolic flux analysis. Additionally, we will perform sequencing of gene variants known to affect platelet function and hormone receptors. Finally, existing microfluidic models of vascular injury will be refined to incorporate endothelial cells to measure platelet-endothelium interactions. Under the second aim, we will use the tools developed in the first aim to measure changes in platelet function following acute and chronic exposure to exogenous hormones. These studies will include tracking platelet function in a cohort of women prior to and after starting OC. The approach is innovative because it represents a new and substantive departure from the status quo by using a systems biology approach to measure and model the influence of sex hormones on platelet function over time scales of seconds to years. The proposed research is significant, because it will identify the mechanism(s) by which exogenous sex hormone confer a pro- and/or antiplatelet phenotype in premenopausal women by both non-genomic and genomic pathways. Ultimately, such knowledge has the potential to improve the safety of hormone therapies in the United States.

Project Summary: In small vessel stroke (SVS), which accounts for 20% of ischemic strokes, tissue plasminogen activator (tPA) is ineffective because it can take a prohibitively long time to diffuse to the clot, and catheter-based thrombectomy devices cannot access small vessels. Moreover, treatment associated hemorrhaging limits tPA use to within a few hours of the onset of symptoms for all ischemic strokes. As a result, there is an urgent need for strategies that overcome these limitations, particularly in SVS, while reducing the risks associated with tPA. Building on a successful previous work, a drug delivery strategy is proposed that can selectively target small artery occlusions and deliver mechanical force to accelerate thrombolysis. The objective of this proposal is to investigate and test within realistic models an approach where injected, dispersed magnetic beads are assembled into blood cell sized microwheels (µwheels) capable of targeting occlusive clots located in small vessels and lysing them with a combination of mechanical and biochemical action. The central hypothesis is that µwheels can (i) target occluded small arteries by exploiting the low flow regions at the entrance of these vessels, (ii) achieve reperfusion at rates an order-of-magnitude faster than soluble tPA, and (iii) improve outcomes in murine models of stroke. This hypothesis will be tested with the following specific aims: Aim 1. Identify magnetic field conditions for µwheels targeting of occlusions. µWheels will be assembled in flowing blood and directed to occluded channels or vessels. Microfluidic, zebrafish, and 3D human cerebrovascular models will be used to test the assembly and targeting. Aim 2. Determine rates for thrombolysis of occlusive thrombi using tPA functionalized µwheels. It is postulated that tPA functionalized µwheels can dissolve fibrin- and platelet-rich clots within microfluidic models and achieve reperfusion in zebrafish and 3D human cerebrovascular models, at rates significantly faster than soluble tPA. Aim 3. Measure the functional benefit of µwheel thrombolysis in vivo. In comparison to soluble tPA, µwheel mediated thrombolysis will improve safety, motor, and neurological outcomes in murine stroke models and can be visualized using high-resolution MRI and micro-CT. In Aims 1 and 2 the expected outcomes are identifying the operating conditions for µwheel assembly, targeting, and fibrinolysis that provide faster reperfusion compared to tPA and can be scaled-up to human-size vascular networks. In Aim 3, it will be shown that µwheel thrombolysis is a superior strategy to systemic administration of tPA in terms of neurobehavioral outcomes in a stroke model and can be imaged in vivo. This approach is significant because it could lead to the development of a more rapid and less invasive strategy for alleviating ischemia than methods currently available. This approach is innovative because of the use of external magnetic fields to propel fibrinolytic microdevices to the sites of occlusion and provide mechanical action to accelerate reperfusion time compared to systemic administration of tPA.

Project Summary: In cystic fibrosis (CF), treatment is difficult because chronic lung infections lead to biofilm formation and drug-resistant bacterial strains prevent antibiotics from working effectively. Antibiotic approaches specifically designed to address infections in the lung include inhaled antibiotics; however, resistant strains are a significant challenge. For delivery, such inhaled drugs must be formulated within a specified size range. Too large and they do not remain suspended to reach deep within the lungs. Too small and they remain in the air and are simply exhaled without embedding. Our scientific premise is that individual magnetic particles of this optimal size range can be inhaled into the lungs and subsequently assembled in place in the form of wheel-like assemblies, or µwheels, to travel deep down lung pathways and disrupt mucus layers to enhance drug-induced biofilm removal. As both µwheel assembly and driving forces are provided by an external magnetic field, once the procedure is finished, devices ?self- disassemble? into small building blocks removable by the body's natural mechanism for removal of dust and other foreign particles in the mucus lining. Our aims include: Aim 1: Identify applied magnetic field conditions that promote µwheel-enhanced biofilm degradation. We will study model P. aeruginosa and CF patient-derived biofilms, with and without artificial sputum, and attach antibiotics or dispersal agents onto the magnetic particle surface and within tortuous microenvironments. We will also use nanoparticle-decorated µwheels to perforate and penetrate the film and test with antibiotic/dispersal agent in solution. Aim 2: Determine conditions that support airborne delivery and transport of µwheels in 3D environments. We will demonstrate airborne delivery of µwheels and translation within 3D models of patient respiratory systems, with and without artificial sputum.

Individuals with hemophilia or taking anticoagulants are at risk for bleeding, but where they bleed is different. Understanding how these two types of perturbations to the hemostatic system interact in distinct vascular beds (VBs) will inform decisions about bleeding treatment. Bleeding is treated using prohemostatic agents, but individual responses to these agents are highly variable and the mechanisms underlying the variability are unknown. Hemostasis is a nonlinear process involving complex coagulation biochemistry coupled to platelet function, VBs, and biophysical mechanisms including blood flow; it is well suited for study with an integrated computational and experimental approach. The long-term goal of this research is to develop mathematical models that improve the treatment of bleeding. The overall objective is to develop and validate mathematical models of bleeding that will identify mechanisms underlying variable responses to prohemostatics and in different VBs. The central hypothesis is that global sensitivity analysis (GSA) applied to mechanistic mathematical models of bleeding will elucidate synergies and/or cooperation among platelet, vascular, and plasma components and predict experimentally-verified hemostatic responses. This hypothesis is based on preliminary data produced using exactly this approach in the applicants? laboratories. The rationale is that the proposed quantitative methods and the identification of modifiers of the hemostatic response will together provide a foundation for developing assays that test for specific and previously unidentified biomarkers. Guided by strong preliminary data, this hypothesis will be tested in three specific aims: 1) Develop and refine mathematical models of hemostasis, 2) Determine the mechanistic link between bleeding site and bleeding cause, and 3) Identify modifiers of hemostasis that regulate responses to prohemostatics in hemophilia A. In Aim 1, existing models will be extended to include essential features of platelet and fibrin dynamics and validated with microfluidic assays. In Aim 2, submodels of anticoagulants will be developed and incorporated into the hemostasis models. Experimental measurements of VB characteristics will be acquired. GSA will identify the causes of VB site-specific variability in the hemostatic response. In Aim 3, submodels of prohemostatics will be developed and incorporated into the hemostasis models. GSA will identify the causes of variability in responses to them during treatment of hemophilia A. The approach is innovative because (1) the mathematical models and experimental assays will be developed in tandem to iteratively and optimally inform one another, and (2) novel submodels of anticoagulants and prohemostatics will be added to a comprehensive model of the hemostatic system that includes platelet, fibrin, and VB dynamics coupled to coagulation and flow. The proposed research is significant because it is expected to (1) provide mechanistic explanations for site- specific bleeding in hemophilia A and anticoagulant use, and (2) provide mechanism-based knowledge to potentially guide clinical decisions in the treatment of bleeding.

PROJECT SUMMARY A critical challenge laid out in the NHLBI Strategic Vision is the development of single-cell analytics to assess health and resiliency. An area where single-cell methods are lacking is in the measurement of platelet reactivity. Specifically, the link between structural heterogeneity and function heterogeneity in platelets is poorly defined. This is a problem because platelets are regulators of bleeding, thrombotic, and inflammatory disorders, and we lack markers of resilience against these disorders. The long-term goal of this line of research is to develop technology that can reveal the mechanistic links between structural and functional heterogeneity in blood cells. The overall objective of this proposal, which is the first step along this continuum of research, is to develop an analytical approach to measure the relationships between mitochondria mass and RNA content and agonist-induced activation at the single platelet level and use that approach to measure changes from individuals from young to old age. Additionally, this approach should allow for pulsatile presentation of agonists because studies in other cells have shown pulsatile stimuli can synchronize response, better identifying subpopulations, and detect low and high frequency band pass filters in signal transduction. The rationale for this approach is that most clinical assays of platelet function measure ensembles of platelets that are unable to discriminate the role of different platelet subpopulations, nor are they able to tie structure and function at the single cell level. We will meet our overall objective with two specific aims: 1) Fabricate and test encapsulated platelets in cast (EPIC) hydrogels to quantify single cell structural and functional heterogeneity; and 2) Measure changes in platelet structure and function with age. We will develop a hydrogel-based platform to rapidly encapsulate fresh platelet isolates and measure the number of organelles or RNA content and correlate these measures with intracellular calcium dynamics and activation markers in response to pulsatile presentation of agonist(s). Using the EPIC platform, we will measure platelet structure and functional changes in platelet from childhood to old age in both sexes. We will test the hypothesis that mitochondrial mass increases with age and correlates with platelet hyperactivity. These studies will be compared to a comprehensive set of platelet phenotyping assays including platelet aggregometry, microfluidic flow assays, flow cytometry, and bioenergetics. The proposed research is innovative, in our opinion, because it represents a substantive departure from the status quo by developing a platform for studying thousands of single platelets in response to dynamic (temporally-modulated) stimuli in minutes. These contributions will be significant because they are expected to provide the means to study normal biological function in platelets in terms of their ability to sense, integrate, and respond to environmental stimuli.