John W. Weisel, Ph.D. - US grants

DESCRIPTION (provided by applicant): The overall goal of this project is to determine specific interactions in the polymerization of fibrin and mechanical stabilization of the clot through network formation and Factor Xllla-catalyzed crosslinking. Research on fibrin polymerization has reached a critical stage at which we know some basic aspects, but fundamental molecular mechanisms remain a mystery. These interactions are difficult or impossible to study by conventional biochemical methods because fibrin is insoluble, many interactions are occurring simultaneously on each molecule, and there is a heterogeneous mixture of species. In the first specific aim, novel techniques we have developed using laser tweezers-based force spectroscopy will be used to study the intermolecular interactions at the single molecule level, so we can separate out and quantify these different binding sites. Mutant fibrinogens with specific impaired binding sites will be used, in addition to fibrin(ogen) fragments. For the second specific aim, deconvolution microscopy, which allows optical sectioning with low fading of fluorescence, will be used to visualize polymerization as a function of time. We will characterize little-known aspects of clot formation, the formation of a branched network, lateral aggregation, and the mechanical stabilization of the network. During the plateau phase of polymerization, fluorescence recovery after photobleaching will be used to measure the remodeling of fibrin. Turnover will be modulated by peptides to compete with the binding interactions and ultrasound. For the third specific aim, the specific interactions of Factor XIII with fibrin(ogen) will be studied by measuring the rupture forces of the individual bonds between Factor XIII or Xllla and fibrinogen or fibrin, as well as variant and mutant molecules. The effects of specific crosslinks on clot properties will be determined. The results of these studies will help us to understand molecular mechanisms of clot formation and stabilization, which may have clinical implications for the treatment and prevention of thrombotic and hemostatic disorders.

Much research on complex biological systems at all levels of structure shares the need for rapid calculation of three dimensional structures and their visualization and manipulation in real time. Structural data at the molecular and cellular levels of the core user group are derived primarily from electron microscopy, while images of living cells are obtained by fluorescent light microscopy; data at the organ and tissue level are derived from living people by the techniques of computed tomography (CT) and magnetic resonance imaging (MRI). The VAXserver 3900, X-windows terminals as well as the Stardent supergraphics computer with enhancements are needed by all users of the facility for computation and visualization of data. The Optronics interface is needed for digitizing data on the existing Optronics microdensitometer. The fluorescent light microscope workstation and CCD and SIT cameras and are necessary for collecting data from living cells. The core user group consists of 14 scientists from six different departments or institutes pursuing ten research projects. The subjects studied by the major users are diverse: muscle thick filament structure, including the backbone and crossbridge interactions, dynamics of development and other changes in muscle and non-muscle cells, excitation-contraction coupling, structure and function of retinal circuitry, role of oncogenes in growth and differentiation, alveolar fluid uptake, fibrinogen and fibrin structure and assembly, and imaging of anatomical structures by CT and MRI. Most of the core users have some experience with computer image processing and graphics applied to biological problems and have research programs that have been limited by the currently available instrumentation. The requested equipment will open exciting new research possibilities for a wide variety of scientists at the University of Pennsylvania.

This proposal requests funds for the purchase of equipment necessary to establish a multi-user Laser Tweezers and Laser Scissors center. The development of optical trapping by laser light using single- beam gradient force traps (popularly referred to as optical tweezers) has provided a powerful tool for micromanipulating cells and polymers both in vivo and in vltro. The instrumentation can be used to elucidate qualitative and quantitative properties of organelles or intracellular bacteria inside living cells as well as particles or bacteria attached to the surfaces of living cells. Experiments are also proposed for the use of the Laser Tweezers to measure the force development of beads coated with motor molecules in cell-free systems. The power of the Laser Scissors can be used to make precise surgical cuts inside living cells as well as in isolated polymer systems. Experiments are also proposed to use the Laser Scissors to cut parts of cells and axons that can then be isolated with the Cell Selector microsyringe so that they can be analyzed for different types of mRNA. The Laser Tweezers and Laser Scissors will be mounted on a requested Nikon inverted microscope. This laser center will be administered by experienced investigators and will be available for investigators and their students at the University of Pennsylvania. The instrumentation will be unique on the campus and will permit problems of a fundamental nature to be addressed that would otherwise not be possible. ~ - I

DESCRIPTION (provided by applicant): A new field of biomedical research, biomechanics of hemostasis and thrombosis, has been quickly developing over the past few years. The mechanical properties of fibrin are naturally variable and largely determine whether clots stanch bleeding, or lead to thrombosis or hemorrhage, and this makes them a desirable therapeutic target. In this application, fibrin mechanics will be studied with respect to structural changes during physiologically relevant fibrin deformations at increasing levels of complexity, including individual molecules, fibrin oligomers, and whole fibrin clots as well as ex vivo thrombi. The structural basis of the viscoelastic properties of fibrin is going to be examined using a uniquely broad, integrated approach based on state-of-the- art biophysical techniques, such as single-molecule optical trap-based force spectroscopy, wide angle X-ray scattering, Fourier Transform infrared spectroscopy, high-resolution rheometry, atomic force microscopy, confocal and electron microscopy, combined with computational molecular dynamics simulations and multiscale modeling. In Specific Aim 1, the structural transitions in fibrin at the molecular level induced by mechanical force will be studied. Understanding of the unfolding of the coiled-coils, ?C regions, and ?-nodules will define the molecular changes that occur in vivo as a result of blood flow, clot retraction, and wound stretching. The ?-helix to ?-sheet transition in the coiled-coils is an important mechanism of fibrin mechanics and potentially tunable for clinical purposes. Straightening of the ?C polymers and unfolding of the ?-nodules also play major roles in fibrin mechanical properties. In Specific Aim 2, nanomechanics of the A:a knob-hole bonds that hold fibrin together will be studied at the single-molecule level. Preliminary data show that at the A:a bonds exhibit counterintuitive catch bond behavior, meaning that the strength of the bond increases with increasing force. This novel finding is a basis for further in depth studies because of its general importance for the field of biomolecular interactions and potential physiological significance. Using a new approach, Binding- Unbinding Correlation Spectroscopy, that we developed we will extensively characterize the two-dimensional kinetics and thermodynamics of formation and dissociation of single A:a bonds. In Specific Aim 3, mechanical properties of clinically significant clots and thrombi will be studied, with a logical progression from the molecular and microscopic levels to increasingly complex macroscopic structures formed in vivo. Screening of chemicals and structural modifications that potentially stabilize or destabilize fibrin molecular domains will be performed to reveal potential modulators of fibrin mechanical properties for therapeutic purposes. These studies would advance the field of hemostasis and thrombosis by leading to new structure- and mechanics-based approaches to prevent and treat bleeding and thrombosis.

? DESCRIPTION (provided by applicant): Surgery increases the risk of thrombosis but prevention and management of thromboembolic disease in the perioperative period is complicated by the risk of hemorrhage. Neutrophils (PMNs) play a vital role in wound healing, but their contribution to perioperative thrombosis and implications for thromboprophylaxis are now emerging. Adherence of PMNs to wounds and to the vasculature generates a unique localized sequestrum enriched in enzymes and antimicrobial peptides protected from plasma inhibitors that contribute to innate immunity in part through the orderly formation and dissolution of fibrin. We posit that post-operative and persistent inflammation disrupt this balance, predisposing to thrombosis by promoting formation and persistence of fibrin. We have previously observed that a-defensins (a-def), antimicrobial peptides that constitute 5% of total human PMN protein that are released upon activation, promote clotting and inhibit fibrinolysis. The absence of a-def in murine PMNs has hindered a better understanding of how these peptides contribute to thromboembolic disease in the perioperative setting. Using a novel transgenic mouse that expresses PMN a-def (Def++), we show that a-def circulates in a complex with fibrinogen and promotes polymerization and retraction of fibrin in vitro, deposits in the vasculature, induces occlusive arterial and venous thrombosis, and inhibits lysis of pulmonary emboli in vivo. These pathogenic properties can be transferred to wild type animals by transplanting bone marrow from Def++ mice and can be prevented and reversed by immunodepleting PMNs or by inhibiting a-def release using colchicine. We now propose an integrated approach to understanding the mechanism and implications of PMN a-def on perioperative thrombosis by examining: a) The biophysical effects of a-def on clot formation and structure; b) The mechanism of accelerated thrombosis and impaired fibrinolysis in Def++ mice and the salutary effect of blocking a-def release; and c) The utility of a-def expression as a biomarker for risk of venous thromboembolism post- surgery. These studies will provide insight into an unappreciated contribution of PMN a-def to arterial and venous thrombosis, identify novel genetic and protein biomarkers of patients at risk for surgery related thrombosis and provide evidence for the use of a safe and effective intervention to prevent thrombosis in the perioperative period.

DESCRIPTION (provided by applicant): NIH-funded researchers at the University of Pennsylvania request funds for a shared instrumentation grant to replace an 18-year old scanning electron microscope (SEM) that has been a workhorse for the School of Medicine (SOM). Despite the best efforts and significant investment of both our faculty and staff and the microscope company, this only SEM available to Penn Medicine researchers has been unreliable for the past few years and has recently become practically unusable. The aim of this proposal is to provide a FEI Quanta 250 microscope that will serve the basic needs for the examination of traditional dehydrated, metal coated samples with SEM, but also including the capability for correlative light microscopy-SEM, imaging of hydrated samples, and back-scattered electron detection of colloidal gold particles. This instrument will support a wide range of research throughout the SOM and across the campus. The studies described here require the substantially enhanced imaging capabilities of this system for NIH-funded research projects. Specifically, all investigators use various types of light microscopy in their research and require the ability to correlate the images obtained in this manner with higher resolution SEM images. Most of the scientists also require the identification and localization of specific macromolecules on the surface of cells or other structures, which requires a back-scattered electron detector. Because of the well-known artifacts of dehydration and metal coating, the ability to image hydrated specimens by environmental SEM is now necessary. We expect that demand and usage for the SEM will continue to rise as a consequence of these new capabilities. An indication of the very strong and broad support for this SEM is the commitment of substantial matching funds from the Department of Cell and Developmental Biology, together with the inclusion of the instrument in the Electron Microscopy Resource Laboratory, with core support and administration by the Department of Biochemistry and Biophysics, and the recognition and support of the SOM administration that SEM is a strategic necessity. This new microscope will allow fundamental biomedical problems to be addressed that would otherwise not be possible, will ensure continued productivity from the researchers, and will maintain the microscopy-training mission of our core facilities.

Non-technical: These Collaborative awards by the Biomaterials program in the Division of Materials Research to University of Pennsylvania and University of Massachusetts at Lowell are to investigate the mechanical behavior of the structural component of blood clots, the fibrin gel. Long-term goal project is to modify fibrin to create distinctive new biomaterials for cell and drug delivery, patterning, and tissue engineering. With this award, the researchers will study the mechanical properties of fibrin clots at the macroscopic, microscopic and submolecular levels to understand the basis of their functional behavior. A key novelty of this work is combined state-of-the-art experimental and theoretical exploration to understand the mechanical behavior of fibrin networks at these different spatial scales. This research will provide opportunities for training of post-doctoral, graduate, and undergraduate students in the highly interdisciplinary areas of nanotechnology and biophysics, with attention to broadening the participation of underrepresented groups. The scientific community will be reached by organizing symposia in international conferences, and by presenting research findings regularly, as well as the NSF-funded Nano/Bio Interface Center for NanoDay and other activities targeted at minority students and teachers in nearby high schools.

Technical: The research objective of this proposal is to understand the mechanical behavior of fibrin gels with the long-term goal of modifying fibrin to create distinctive new biomaterials for cell and drug delivery, patterning, and tissue engineering. Earlier studies by the researchers have demonstrated that the remarkable extensibility of fibrin clots has its origins in the unfolding of particular domains of the fibrin molecule, with the stress-strain response of an individual fiber connected to the force-stretch dependence at the nanoscale. Researchers plan to define the molecular structural and thermodynamic basis of fibrin deformability and viscoelasticity. A key novelty of this work is combined experimental and theoretical exploration to understand the tensile behavior of fibrin networks at different spatial scales, from submolecular (atomic) to macroscopic levels. This goal will be achieved by combining state-of-the-art experimental methodology with novel Molecular Dynamics simulations and a comprehensive continuum mechanics model. This research will provide opportunities for training of post-doctoral, graduate, and undergraduate students in the highly interdisciplinary areas of nanotechnology and biophysics, with attention to broadening the participation of underrepresented groups. The scientific findings will be disseminated by organizing symposia in both national and international levels. The research findings will be presented to the community by attending other scientific meetings, as well as the NSF-funded Nano/Bio Interface Center for NanoDay and other activities targeting minority students and teachers in high schools.