Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 ± 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 ± 4.1 μm, n = 63) were pulled at an average rate of 6–40 μm/s as the wall shear rate was increased from 100–250 s−1. The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s−1 (Fbond = 86 pN) to 250 s−1 (Fbond = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s−1, thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 ± 8.8 μm long), whereas tether breakage (average lifetime of 3.79 ± 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 μm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil's surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)(2)Cl(2+/3+)] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to approximately 1 mA/cm(2) for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.

Hydrogen peroxide sensing microelectrodes for scanning electrochemical microscopy (SECM) have been developed and used to measure hydrogen peroxide in the diffusion layer during electrochemical reduction of oxygen on gold and carbon electrodes. These microelectrode biosensors were also used to detect immobilized glucose oxidase through the production of hydrogen peroxide during enzymatic oxidation of glucose. Images of hydrogen peroxide concentration profiles near a platinum microdisk during catalytic decomposition of peroxide and in the diffusion layer of a carbon-platinum composite electrode during oxygen reduction are presented. The factors limiting the spatial resolution (tens of micrometers) and potential applications of the technique are discussed.

In inflamed venules, leukocytes use Pselectin glycoprotein ligand-1 (PSGL-1) to roll on P-selectin and E-selectin and to activate integrin ␣L␤2 (lymphocyte function-associated antigen-1, LFA-1) to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Studies in cell lines have suggested that PSGL-1 requires its cytoplasmic domain to localize in membrane domains, to support rolling on Pselectin, and to signal through spleen tyrosine kinase (Syk). We generated "⌬CD" mice that express PSGL-1 without the cytoplasmic domain. Unexpectedly, neutrophils from these mice localized PSGL-1 normally in microvilli, uropods, and lipid rafts. ⌬CD neutrophils expressed less PSGL-1 on their surfaces because of inefficient export from the endoplasmic reticulum. Limited digestion of wild-type neutrophils with O-sialoglycoprotein endopeptidase was used to reduce the PSGL-1 density to that on ⌬CD neutrophils. At matched PSGL-1 densities, both ⌬CD and wild-type neutrophils rolled similarly on P-selectin. However, ⌬CD neutrophils rolling on P-selectin did not trigger Sykdependent activation of LFA-1 to slow rolling on ICAM-1. These data demonstrate that the PSGL-1 cytoplasmic domain is dispensable for leukocyte rolling on P-selectin but is essential to activate ␤2 integrins to slow rolling on ICAM-1. IntroductionDuring inflammation, leukocytes tether to and roll on the vessel wall. They then roll more slowly until they arrest. Finally, they crawl through or between endothelial cells into the underlying tissues. Interactions of selectins with glycosylated ligands mediate tethering and rolling. Interactions of ␤2 integrins with ligands, such as intercellular adhesion molecule-1 (ICAM-1), mediate slow rolling and arrest. 1,2 These interactions occur in blood flow, which exerts force on adhesive bonds that affects bond lifetimes. 3,4 Furthermore, engagement of adhesion receptors transmits signals that intersect with chemokine receptor signals to influence the adhesion cascade. 1 Binding of integrin cytoplasmic domains to signaling and cytoskeletal proteins is critical for integrin function. 1,5 Interactions of selectin cytoplasmic domains with cytosolic proteins also contribute to their adhesive properties. E-selectin and P-selectin are expressed on activated endothelial cells and/or platelets, whereas L-selectin is expressed on the microvilli of leukocytes. 2 The cytoplasmic domains of E-selectin and P-selectin interact with clathrin-coated pits. These interactions cluster E-selectin and P-selectin on the endothelial cell surface, enhancing leukocyte rolling under flow. 6,7 The cytoplasmic domain anchors L-selectin to the cytoskeleton by binding to ␣-actinin 8 and to the ezrin/radixin/ moesin (ERM) family. 9 Mutation of the ERM-binding site in the cytoplasmic domain shifts L-selectin out of microvilli onto the cell body of transfected cells and impairs tethering to L-selectin ligands under flow. 10 Removal of the ␣-actinin-binding site markedly impairs rolling of transfected cells on L-selectin ligands, and deletion of the cytoplas...