Hammer, Daniel A

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2000 - 200825
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  • Publication
    Effect of Shear Stress on Platelet Activation via the Glycoprotein VI Receptor
    (2002-10-23) Sarratt, Kendra L.; Chen, Hong M.; Kahn, Mark L.; Hammer, Daniel A
    Cardiovascular diseases are the nation's leading cause of death. Such diseases are caused by platelet response to collagen especially in the event of vascular injury leading to thrombosis. One of the platelet receptors known to bind to the collagen ligand is glycoprotein VI (GPVI) with co-receptor Fc receptor γ chain (FcRγ). By stably expressing the GPVI receptor in rat basophilic leukemia cells (RBL-2H3), which abundantly express FcRγ, but endogenously lack GPVI, studies have shown that GPVI-FcRγ is sufficient to confer adhesion as well as signaling responses to collagen as long as the receptor density is equivalent to that found on human platelets. While those investigations confirm that the GPVI receptor mediate binding to collagen under static conditions, they do not provide information on how the GPVI receptor interacts with collagen under dynamic conditions. In the present study we have used the GPVI-expressing RBL-2H3 cells to observe the kinetics of adhesion to collagen under hydrodynamic flow conditions in vitro using a parallel plate flow chamber coupled with video microscopy. We demonstrate that these cells do adhere to the surface at a low shear rate and do so at a greater adherent cell density than wild-type RBL-2H3 (WT-RBL) cells.
  • Publication
    A Microcantilever Device to Assess the Effect of Force on the Lifetime of Selectin-Carbohydrate Bonds
    (2001-02-01) Tees, Davis F. J.; Waugh, Richard E; Hammer, Daniel A
    A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-μm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewisx, was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), rf. From the distribution of forces at failure, the average force was determined and plotted as a function of ln rf. The slope and intercept of the plot yield the unstressed reverse reaction rate, kro , and a parameter that describes the force dependence of reverse reaction rates, ro. The ligand was titrated so adhesion occurred in ~30% of tests, implying that >80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are ro = 0.048 and 0.016 nm and kro = 0.72 and 2.2 s-1 for loading rates in the ranges 200–1000 and 1000–5000 pN s-1, respectively. Levenberg-Marquardt fitting across all values of rf gives ro = 0.034 nm and kro = 0.82 s-1. The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations.
  • Publication
    Rolling Adhesion of Yeast Engineered to Express Cell Adhesion Molecules
    (2002-04-20) Bhatia, Sujata K; Swers, Jeffrey S.; Camphausen, Raymond T.; Hammer, Daniel A; Wittrup, K. Dane
    Selectins are cell adhesion molecules that mediate capture of leukocytes on vascular endothelium as an essential component of the inflammatory response. Here we describe a method for yeast surface display of selectins, together with a functional assay that measures rolling adhesion of selectin-expressing yeast on a ligand-coated surface. E-selectin-expressing yeast roll specifically on surfaces bearing sialyl-Lewisx ligands. Observation of yeast rolling dynamics at various stages of their life cycle indicates that the kinematics of yeast motion depends on the ratio of the bud radius to the parent radius (B/P). Large-budded yeast "walk" across the surface, alternately pivoting about bud and parent. Small-budded yeast "wobble" across the surface, with bud pivoting about parent. Tracking the bud location of budding yeast allows measurement of the angular velocity of the yeast particle. Comparison of translational and angular velocities of budding yeast demonstrates that selectin-expressing cells are rolling rather than slipping across ligand-coated surfaces.
  • Publication
    The Shear Threshold Effect for Particle Adhesion to Bioreactive Surfaces: Influence of Receptor and Ligand Site Density
    (2003-03-22) Bhatia, Sujata K.; Hammer, Daniel A
    Selectins are cell adhesion molecules that mediate capture and rolling adhesion of white blood cells to vascular walls, an essential component of the inflammatory response. Adhesion through L-selectin requires a hydrodynamic shear stress above a threshold level, a phenomenon known as the shear threshold effect. We have reported that the shear threshold effect can he re-created in cell-free systems, in which microspheres coated with the carbohydrate ligand sialyl Lewis x (sLex) are perfused over L-selectin-coated surfaces. This paper extends the use of the cell-free system to determine the concurrent influence of receptor and ligand site density on the shear threshold effect. We find that the shear threshold effect diminishes with increasing levels of either L-selectin or sLex. At reduced site densities of either L-selectin or sLex, the shear threshold effect is present, with maximal rolling observed at a shear stress of 1.2 dynes/cm2. At higher site densities of L-selectin and sLex, the shear threshold effect disappears. These results suggest that the shear threshold relies on the formation of low numbers of receptor-ligand bonds.
  • Publication
    Quantitative Membrane Loading of Polymer Vesicles
    (2006-11-07) Ghoroghchian, P. Peter; Lin, John J; Brannon, Aaron K; Frail, Paul R; Therien, Michael J; Bates, Frank S; Hammer, Daniel A
    We utilize a series of structurally homologous, multi-porphyrin-based, fluorophores (PBFs) in order to explore the capacity of polymer vesicles (polymersomes) to stably incorporate large hydrophobic molecules, non-covalently within their thick lamellar membranes. Through aqueous hydration of dry, uniform thin-films of amphiphilic polymer and PBF species deposited on Teflon, self-assembled polymersomes are readily generated incorporating the hydrophobic fluorophores in prescribed molar ratios within their membranes. The size-dependent spectral properties of the PBFs allow for ready optical verification (via steady-state absorption and emission spectroscopy) of the extent of vesicle membrane loading and enable delineation of intermembranous molecular interactions. The resultant effects of PBF membrane-loading on polymersome thermodynamic and mechanical stability are further assessed by cryogenic transmission electron microscopy (cryo-TEM) and micropipet aspiration, respectively. We demonstrate that polymersomes can be loaded at up to 10 mol/wt% concentrations, with hydrophobic molecules that possess sizes comparable to those of large pharmaceutical conjugates (e.g. ranging 1.4–5.4 nm in length and Mw = 0.7–5.4 kg mol–1), without significantly compromising the robust thermodynamic and mechanical stabilities of these synthetic vesicle assemblies. Due to membrane incorporation, hydrophobic encapsulants are effectively prevented from self-aggregation, able to be highly concentrated in aqueous solution, and successfully shielded from deleterious environmental interactions. Together, these studies present a generalized paradigm for the generation of complex multi-functional materials that combine both hydrophilic and hydrophobic agents, in mesoscopic dimensions, through cooperative self-assembly.
  • Publication
    A Semianalytic Model of Leukocyte Rolling
    (2004-11-01) Krasik, Ellen F; Hammer, Daniel A
    Rolling allows leukocytes to maintain adhesion to vascular endothelium and to molecularly coated surfaces in flow chambers. Using insights from adhesive dynamics, a computational method for simulating leukocyte rolling and firm adhesion, we have developed a semianalytic model for the steady-state rolling of a leukocyte. After formation in a force-free region of the contact zone, receptor-ligand bonds are transported into the trailing edge of the contact zone. Rolling velocity results from a balance of the convective flux of bonds and the rate of dissociation at the back edge of the contact zone. We compare the model’s results to that of adhesive dynamics and to experimental data on the rolling of leukocytes, with good agreement. We calculate the dependence of rolling velocity on shear rate, intrinsic forward and reverse reaction rates, bond stiffness, and reactive compliance, and use the model to calculate a state diagram relating molecular parameters and the dynamic state of adhesion. A dimensionless form of the analytic model permits exploration of the parameters that control rolling. The chemical affinity of a receptor-ligand pair does not uniquely determine rolling velocity. We elucidate a fundamental relationship between off-rate, ligand density, and reactive compliance at the transition between firm and rolling adhesion. The model provides a rapid method for screening system parameters for the potential to mediate rolling.
  • Publication
    Cell-Free Rolling Mediated by L-Selectin and Sialyl Lewisx Reveals the Shear Threshold Effect
    (2000-11-01) Greenberg, Adam W; Brunk, Debra K; Hammer, Daniel A
    The selectin family of adhesion molecules mediates attachment and rolling of neutrophils to stimulated endothelial cells. This step of the inflammatory response is a prerequisite to firm attachment and extravasation. We have reported that microspheres coated with sialyl Lewisx (sLex) interact specifically and roll over E-selectin and P-selectin substrates (Brunk et al., 1996; Rodgers et al., 2000). This paper extends the use of the cell-free system to the study of the interactions between L-selectin and sLex under flow. We find that sLex microspheres specifically interact with and roll on L-selectin substrates. Rolling velocity increases with wall shear stress and decreases with increasing L-selectin density. Rolling velocities are fast, between 25 and 225 μm/s, typical of L-selectin interactions. The variability of rolling velocity, quantified by the variance in rolling velocity, scales linearly with rolling velocity. Rolling flux varies with both wall shear stress and L-selectin site density. At a density of L-selectin of 800 sites/μm2, the rolling flux of sLex coated microspheres goes through a clear maximum with respect to shear stress at 0.7 dyne/cm2. This behavior, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, is known as the shear threshold effect. We found that the magnitude of the effect is greatest at an L-selectin density of 800 sites/μm2 and gradually diminishes with increasing L-selectin site density. Our study is the first to reveal the shear threshold effect with a cell free system and the first to show the dependence of the shear threshold effect on L-selectin site density in a reconstituted system. Our ability to recreate the shear threshold effect in a cell-free system strongly suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and largely eliminates cellular features such as deformability or topography as its cause.
  • Publication
    The Effect of Cellular Receptor Diffusion on Receptor-Mediated Viral Binding Using Brownian Adhesive Dynamics (BRAD) Simulations
    (2005-03-01) English, Thomas J.; Hammer, Daniel A
    Brownian adhesive dynamics (BRAD) is a new method for simulating the attachment of viruses to cell surfaces. In BRAD, the motion of the virus is subject to stochastic bond formation and breakage, and thermal motion owing to collisions from the solvent. In the model, the virus is approximated as a rigid sphere and the cell surface is approximated as a rigid plane coated with receptors. In this article, we extend BRAD to allow for the mobility of receptors in the plane of the membrane, both before and after they are ligated by viral attachment proteins. Allowing the proteins to move within the membrane produced several differences in behavior from when the receptors are immobilized. First, the mean steady-state bond number is unaffected by changes in cellular receptor density because proteins are now free to diffuse into the contact area, and the extent of binding is dictated by the availability of viral attachment proteins. Second, the time required to reach steady-state binding increases as both the cellular receptor number decreases and the receptor mobility decreases. This is because receptor diffusion is a slower process than the binding kinetics of the proteins. Decreasing the rate of protein binding was found to decrease the fraction of viruses bound to steady state, but not the extent of binding for those viruses that were bound. Increasing the binding rate increased the fraction of viruses bound, until no further viruses could bind. Alterations in receptor binding kinetics had no discernable effect on the mean steady-state bond number between virus and cell, because interactions were of sufficiently high affinity that all available receptor-viral attachment proteins were destined to bind at steady state.
  • Publication
    Adhesive Dynamics Simulations of the Shear Threshold Effect for Leukocytes
    (2008-02-01) Kaputo, Kelly; Lee, Dooyoung; King, Michael R; Hammer, Daniel A
    Many experiments have measured the effect of force on the dissociation of single selectin bonds, but it is not yet clear how the force dependence of molecular dissociation can influence the rolling of cells expressing selectin molecules. Recent experiments using constant-force atomic force microscopy or high-resolution microscopic observations of pause-time distributions of cells in a flow chamber show that for some bonds, the dissociation rate is high at low force and initially decreases with force, indicating a catch bond. As the force continues to increase, the dissociation rate increases again, like a slip bond. It has been proposed that this catch-slip bond leads to the shear threshold effect, in which a certain level of shear rate is required to achieve rolling. We have incorporated a catch-slip dissociation rate into adhesive dynamics simulations of cell rolling. Using a relatively simple model for the shear-controlled association rate for selectin bonds, we were able to recreate characteristics of the shear threshold effect seen most prominently for rolling through L-selectin. The rolling velocity as a function of shear rate showed a minimum near 100 s–1. Furthermore, cells were observed to roll at a shear rate near the threshold, but detach and move more quickly when the shear rate was dropped below the threshold. Finally, using adhesive dynamics, we were able to determine ranges of parameters necessary to see the shear threshold effect in the rolling velocity. In summary, we found through simulation that the catch-slip behavior of selectin bonds can be responsible for the shear threshold effect.
  • Publication
    Oligomerization of Fusogenic Peptides Promotes Membrane Fusion by Enhancing Membrane Destabilization
    (2004-01-01) Lear, James D; Lau, Wai Leung; Hammer, Daniel A; Ege, David S; DeGrado, William F
    A key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triplehelical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, τlysis, but not area expansion modulus, Ka, of phospholipid bilayers, whereas monomeric X31 peptide lowers both τlysis and Ka. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers.