Title: Low Force Decelerates L-selectin Dissociation from P-selectin Glycoprotein Ligand-1 and Endoglycan
Abstract: Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions. Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions. Selectins are a family of adhesion molecules that contribute to leukocyte trafficking during inflammation and tissue injury and to lymphocyte homing (1.Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Crossref PubMed Scopus (836) Google Scholar, 2.McEver R.P. Thromb. Haemostasis. 2001; 86: 746-756Crossref PubMed Scopus (367) Google Scholar, 3.McEver R.P. Curr. Opin. Cell Biol. 2002; 14: 581-586Crossref PubMed Scopus (367) Google Scholar). E-selectin is expressed on activated endothelial cells; L-selectin is expressed on leukocytes, and P-selectin is expressed on activated endothelial cells and platelets. L- and P-selectins interact in a related manner with P-selectin glycoprotein ligand-1 (PSGL-1), 1The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; AFM, atomic force microscopy; BSA, bovine serum albumin; DFS, dynamic force spectroscopy; HBSS, Hanks' balanced salt solution; HSA, human serum albumin; mAb, monoclonal antibody; PEI, polyethyleneimine; PZT, piezoelectric translator; pN, piconewman. a mucin expressed on leukocytes. These interactions mediate tethering and rolling of circulating leukocytes on endothelial cells, platelets, and adherent leukocytes under flow. Selectin-ligand interactions are rapid and transient. In addition, the mechanically stressful environment in the circulation imposes forces on selectin-ligand bonds, which affect their dissociation rates. Bell suggested that applied force could accelerate bond dissociation, because work done by the force could lower the energy barrier between the bound and free states (4.Bell G.I. Science. 1978; 200: 618-627Crossref PubMed Scopus (3523) Google Scholar). Dembo et al. (5.Dembo M. Tourney D.C. Saxman K. Hammer D. Proc. R. Soc. Lond. 1988; 234: 55-83Crossref PubMed Scopus (696) Google Scholar, 6.Dembo M. Lectures on Mathematics in the Life Sciences, Some Mathematical Problems in Biology. 24. American Mathematical Society, Providence, RI1994: 51-77Google Scholar) conversely envisioned that force could also decelerate bond dissociation by deforming the molecules such that they locked more tightly. These two types of behavior are named slip and catch bonds, respectively. Bonds whose off-rates are independent of force are called ideal bonds (5.Dembo M. Tourney D.C. Saxman K. Hammer D. Proc. R. Soc. Lond. 1988; 234: 55-83Crossref PubMed Scopus (696) Google Scholar, 6.Dembo M. Lectures on Mathematics in the Life Sciences, Some Mathematical Problems in Biology. 24. American Mathematical Society, Providence, RI1994: 51-77Google Scholar). Since the first experimental determination of the relationship between force and off-rate (7.Alon R. Hammer D.A. Springer T.A. Nature. 1995; 374: 539-542Crossref PubMed Scopus (604) Google Scholar), many studies found slip bond behavior (7.Alon R. Hammer D.A. Springer T.A. Nature. 1995; 374: 539-542Crossref PubMed Scopus (604) Google Scholar, 8.Alon R. Chen S. Puri K.D. Finger E.B. Springer T.A. J. Cell Biol. 1997; 138: 1169-1180Crossref PubMed Scopus (317) Google Scholar, 9.Alon R. Chen S. Fuhlbrigge R. Puri K.D. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11631-11636Crossref PubMed Scopus (104) Google Scholar, 10.Chen S. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 950-955Crossref PubMed Scopus (161) Google Scholar, 11.Evans E. Leung A. Hammer D. Simon S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3784-3789Crossref PubMed Scopus (192) Google Scholar, 12.Pierres A. Benoliel A.M. Bongrand P. van der Merwe P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15114-15118Crossref PubMed Scopus (78) Google Scholar, 13.Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (122) Google Scholar, 14.Ramachandran V. Yago T. Epperson T.K. Kobzdej M.M.A. Nollert M.U. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10166-10171Crossref PubMed Scopus (115) Google Scholar, 15.Smith M.J. Berg E.L. Lawrence M.B. Biophys. J. 1999; 77: 3371-3383Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 16.Dwir O. Kansas G.S. Alon R. J. Cell Biol. 2001; 155: 145-156Crossref PubMed Scopus (90) Google Scholar, 17.Dwir O. Steeber D.A. Schwarz U.S. Camphausen R.T. Kansas G.S. Tedder T.F. Alon R. J. Biol. Chem. 2002; 277: 21130-21139Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18.Yago T. Leppänen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). Counter-intuitive catch bonds were only recently observed for interactions of P-selectin with PSGL-1 in a force range below those measured previously (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar). Both P- and L-selectin bind to an N-terminal region of PSGL-1 that must be modified with tyrosine sulfates and an appropriately oriented, sialylated, and fucosylated O-glycan (3.McEver R.P. Curr. Opin. Cell Biol. 2002; 14: 581-586Crossref PubMed Scopus (367) Google Scholar, 13.Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (122) Google Scholar, 20.Leppänen A. Mehta P. Ouyang Y.-B. Ju T. Helin J. Moore K.L. van Die I. Canfield W.M. McEver R.P. Cummings R.D. J. Biol. Chem. 1999; 274: 24838-24848Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 21.Leppänen A. White S.P. Helin J. McEver R.P. Cummings R.D. J. Biol. Chem. 2000; 275: 39569-39578Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 22.Somers W.S. Tang J. Shaw G.D. Camphausen R.T. Cell. 2000; 103: 467-479Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar, 23.Leppänen A. Yago T. Otto V.I. McEver R.P. Cummings R.D. J. Biol. Chem. 2003; 278: 26391-26400Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Monoclonal antibodies (mAbs) to this region block binding of PSGL-1 to both P- and L-selectin (24.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichen- stein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (633) Google Scholar, 25.Snapp K.R. Ding H. Atkins K. Warnke R. Luscinskas F.W. Kansas G.S. Blood. 1998; 91: 154-164Crossref PubMed Google Scholar). Compared with P-selectin, L-selectin binds to PSGL-1 with lower affinity and more rapid dissociation kinetics (9.Alon R. Chen S. Fuhlbrigge R. Puri K.D. Springer T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11631-11636Crossref PubMed Scopus (104) Google Scholar, 13.Ramachandran V. Nollert M.U. Qiu H. Liu W.J. Cummings R.D. Zhu C. McEver R.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13771-13776Crossref PubMed Scopus (122) Google Scholar, 15.Smith M.J. Berg E.L. Lawrence M.B. Biophys. J. 1999; 77: 3371-3383Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). However, the general binding similarities suggest that the L-selectin-PSGL-1 interaction, like the P-selectin-PSGL-1 interaction, might also behave as catch-slip transitional bonds. Catch bonds have been suggested to partly explain the shear threshold requirement for selectin-mediated adhesion (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar). Below the threshold, fewer cells roll, and those that do roll less stably and with higher velocity (26.Finger E.B. Puri K.D. Alon R. Lawrence M.B. von Andrian U.H. Springer T.A. Nature. 1996; 379: 266-269Crossref PubMed Scopus (413) Google Scholar, 27.Lawrence M.B. Kansas G.S. Kunkel E.J. Ley K. J. Cell Biol. 1997; 136: 717-727Crossref PubMed Scopus (305) Google Scholar). Bond lifetimes at low wall shear stresses might be too brief to support stable rolling. As the wall shear stress is increased, catch bonds might retard dissociation at the trailing edge, thereby stabilizing rolling, increasing the number of rolling cells, and lowering their velocities. Compared with that for P-selectin, the shear threshold of L-selectin-mediated leukocyte rolling is much more pronounced and occurs over a much wider range of wall shear stresses (26.Finger E.B. Puri K.D. Alon R. Lawrence M.B. von Andrian U.H. Springer T.A. Nature. 1996; 379: 266-269Crossref PubMed Scopus (413) Google Scholar, 27.Lawrence M.B. Kansas G.S. Kunkel E.J. Ley K. J. Cell Biol. 1997; 136: 717-727Crossref PubMed Scopus (305) Google Scholar). If catch bonds contribute to the shear threshold, the L-selectin-PSGL-1 interaction should behave as catch-slip transitional bonds with transition over a much wider force range. Endoglycan is a recently identified PSGL-1-like member of the CD34 family of sialomucins expressed on endothelial cells (28.Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Interactions of endoglycan with L-selectin also support cell rolling under flow (29.Fieger C.B. Sassetti C.M. Rosen S.D. J. Biol. Chem. 2003; 278: 27390-27398Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). If catch bonds are a general feature of selectin-ligand interactions, the L-selectin-endoglycan interaction should also exhibit catch-slip transitional bonds. We tested these hypotheses by using atomic force microscopy (AFM) and flow chamber experiments to measure off-rates of L-selectin dissociating from two forms of PSGL-1, from endoglycan, and from an antibody. Catch-slip transitional bonds were documented in interactions of L-selectin with ligands but not with antibody, which prompts renewed interest in how to model these bonds. Proteins and Antibodies—A cDNA encoding the lectin and epidermal growth factor domains and the two consensus repeats of human L-selectin plus a donor splicing sequence was amplified by the PCR and ligated into the pIG1 vector (30.Simmons D.L. Hartley D. Cellular Interactions in Development. Oxford University Press, Oxford1993: 93-128Google Scholar), which encodes the heavy chain CH2-CH3 and hinge region of human IgG1. The vector was transiently transfected into COS7 cells using FuGENE 6 (Roche Applied Science). Cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The L-selectin-Ig was purified from supernatants by protein G affinity chromatography. Membrane L-selectin was purified from human tonsils (27.Lawrence M.B. Kansas G.S. Kunkel E.J. Ley K. J. Cell Biol. 1997; 136: 717-727Crossref PubMed Scopus (305) Google Scholar), and native dimeric PSGL-1 was purified from human neutrophils (31.Moore K.L. Stults N.L. Diaz S. Smith D.F. Cummings R.D. Varki A. McEver R.P. J. Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (425) Google Scholar). Recombinant monomeric soluble PSGL-1 (sPSGL-1) was purified from supernatants of CHO cell transfectants (18.Yago T. Leppänen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). The endoglycan-Ig, which consists of the extracellular region of the molecule fused to a human Fc domain, was collected from transfected COS7 cell supernatants (28.Sassetti C. Van Zante A. Rosen S.D. J. Biol. Chem. 2000; 275: 9001-9010Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The blocking anti-L-selectin mAb DREG56 (32.Kishimoto T.K. Jutila M.A. Butcher E.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2244-2248Crossref PubMed Scopus (397) Google Scholar) and the blocking (PL1) and capture (PL2) anti-PSGL-1 mAbs (24.Moore K.L. Patel K.D. Bruehl R.E. Fugang L. Johnson D.A. Lichen- stein H.S. Cummings R.D. Bainton D.F. McEver R.P. J. Cell Biol. 1995; 128: 661-671Crossref PubMed Scopus (633) Google Scholar) have been described. Anti-human IgG Fc polyclonal antibody was purchased from Chemicon (Temecula, CA). The AFM System—The AFM system was built and calibrated in-house (design adapted from Vincent Moy, University of Miami) (Fig. 1A) (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar). It consists of a piezoelectric translator (PZT) (Polytec Physik Instrument, Boston) on which a cantilever (Thermomicroscopes, Sunnyvale, CA) is directly mounted. A laser (Oz Optics, Ontario, Canada) focused on the end of the back of the cantilever is deflected onto a photodiode (Hamamatsu, Bridgewater, NJ) to measure cantilever deflection, which is converted to force using the cantilever spring constant. Each cantilever spring constant (4-30 pN/nm) was calibrated during the experiment using thermal fluctuation analysis (33.Hutter J.L. Bechhoefer J. Rev. Sci. Instrum. 1993; 64: 1868-1873Crossref Scopus (3519) Google Scholar). A personal computer with a data acquisition board (National Instruments, Austin, TX) was used to control the movement of the PZT and to collect the signal from the photodiode. LabView (National Instruments) was used as the interface between the user and the data acquisition board. Functionalizing the AFM Cantilever Tip—Cantilever tips were incubated overnight at 4 °C in 10 μg/ml PL2, protein G, or DREG56 or 1% bovine serum albumin (BSA). After rinsing, the coated cantilevers were incubated for 30 min at room temperature in Hanks' balanced salt solution (HBSS) containing 1% BSA to block nonspecific adhesion. PL2-coated (or protein G-coated) cantilevers were further functionalized by incubation in 10 μl of 100 ng/ml (s)PSGL-1 (or endoglycan supernatant) for 20 min at room temperature. DREG56-, BSA-, and some PL2- and protein G-coated cantilevers were used without further modification (Fig. 1B). Formation of L-selectin Bilayer—Membrane L-selectin was reconstituted into glass-supported, polyethyleneimine (PEI)-cushioned lipid bilayers (Fig. 1B) by using vesicle fusion (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar, 34.Wong J.Y. Majewski J. Seitz M. Park C.K. Israelachvili J.N. Smith G.S. Biophys. J. 1999; 77: 1445-1457Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 35.McConnell H.M. Watts T.H. Weis R.M. Brian A.A. Biochim. Biophys. Acta. 1986; 864: 95-106Crossref PubMed Scopus (503) Google Scholar, 36.Dustin M.L. Olive D. Springer T.A. J. Exp. Med. 1989; 169: 503-517Crossref PubMed Scopus (79) Google Scholar). Our AFM experiments attempted to measure specific bond lifetimes at forces as low as thermally driven force fluctuations, which have a standard deviation of ∼4-11 pN at room temperature, depending on the stiffness of the cantilever used (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar). These are much smaller than the adhesion forces between coverslips and SiN3 cantilever tips, yielding ∼100% nonspecific binding because all nonspecific interactions can be detected. Covering the glass with an intact lipid bilayer reduced the nonspecific binding to nearly zero. Nonspecific binding was increased when L-selectin was incorporated in the bilayer, presumably because it was partly disrupted by the inversely inserted molecules that have extracellular domains much larger than the normal gap distance between the bilayer and the coverglass. It was crucial to add a PEI cushion to accommodate the inversely oriented molecules between the glass and the bilayer (Fig. 1B), which reduced the nonspecific binding to ∼5% (Fig. 2, A-D). Coverslips of 40 mm in diameter (Bioptechs, Butler, PA) were cleaned by using a mixture of 70% 12 n sulfuric acid and 30% hydrogen peroxide by volume at a temperature of 100 °C for 45 min, rinsed extensively with deionized water, and dried completely under an argon stream. The cleaned coverslips were stored in a desiccator and used within a 2-week period. The coverslip was first immersed in a 100-ppm PEI (Mr = 1800 g/mol, 95% purity; Polysciences, Inc., Warrington, PA) solution of 0.5 mm KNO3 (Fisher) in deionized water, pH 7.0, for 20 min. After rinsing, excess water was removed from the coverslip surface by a gentle stream of argon. The coverslip was then placed in a desiccator for 10 min to dry the PEI layer (34.Wong J.Y. Majewski J. Seitz M. Park C.K. Israelachvili J.N. Smith G.S. Biophys. J. 1999; 77: 1445-1457Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) before forming an L-selectin bilayer on it. An L-selectin-incorporated lipid vesicle solution was prepared following the method of McConnell et al. (35.McConnell H.M. Watts T.H. Weis R.M. Brian A.A. Biochim. Biophys. Acta. 1986; 864: 95-106Crossref PubMed Scopus (503) Google Scholar) and Dustin et al. (36.Dustin M.L. Olive D. Springer T.A. J. Exp. Med. 1989; 169: 503-517Crossref PubMed Scopus (79) Google Scholar). Briefly, vesicles were formed by hydrating a dried lipid film of egg phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) with 2% octyl β-glucopyranoside (Fisher), Tris saline solution creating 250 μl of 0.8 mm lipid solution. The 2% octyl β-glucopyranoside, egg phosphatidylcholine solution was combined with 250 μl of 1% octyl β-glucopyranoside solution with a 28 μg/ml concentration of membrane L-selectin. The resulting 0.4 mm lipid solution was dialyzed with three 1-liter changes of Tris saline buffer (25 mm Tris-HCl, 150 mm NaCl, pH 7.4) in 12-h increments. The resulting lipid vesicle solution was stored under argon at 4 °C and used within several months. Bilayers were formed using the method of vesicle fusion (35.McConnell H.M. Watts T.H. Weis R.M. Brian A.A. Biochim. Biophys. Acta. 1986; 864: 95-106Crossref PubMed Scopus (503) Google Scholar, 36.Dustin M.L. Olive D. Springer T.A. J. Exp. Med. 1989; 169: 503-517Crossref PubMed Scopus (79) Google Scholar). A PEI-coated coverslip was placed in a Petri dish, and a 4-μl drop of L-selectin-incorporated lipid vesicle solution was placed on the surface of the coverslip. After 20 min of incubation under a damp paper towel, the Petri dish was filled with 10 ml of HBSS with 1% BSA. The L-selectin bilayers so formed had low molecular densities, which ensured their infrequent binding to the (s)PSGL-1-, endoglycan-, or DREG56-coated cantilever tips, as required for measuring single bond interactions. Bilayers were immediately used in AFM experiments. AFM Adhesion Event and Lifetime Measurements—Binding was enabled by driving the cantilever tip with the PZT to contact the bilayer for 2 s with an ∼30-pN compressive force. The cantilever was then retracted at a speed of 250 nm/s. The presence or absence of adhesion was detected from the force-scan curves. The curves in Fig. 1, C and D, illustrate how the cantilever was bent (Fig. 1D, insets) when a compressive or a tensile force was respectively applied to the tip. The approach curves were horizontal (zero mean force) initially but were bent downward when the tip was pressed onto the bilayer. The retraction curve mirrored the approach curve until the cantilever was no longer bent. The upper curves in Fig. 1, C and D, illustrate a contact cycle without binding, where the retraction curve returned to zero mean force. When the tip was linked to the bilayer by a molecular bond, continued retraction yielded a tensile force that signified binding, as illustrated by the lower curves in Fig. 1, C and D. Adhesion frequency was measured as the number of binding events per 100 repeated approach-retraction cycles using the same tip contacting the same L-selectin bilayer spot. Once the cantilever retracted a predetermined distance and stopped, a constant force was applied to the bond, if a bond was present, and it survived the ramping to the desired force. The lifetime was measured from the instant when the PZT stopped to the instant of bond failure. Force-scan data were acquired at 5 kHz. Since most of the lifetimes were tens of milliseconds or longer, a number of instantaneous forces were averaged to allow lifetime measurement at forces comparable with the level of thermal fluctuations (19.Marshall B.T. Long M. Piper J.W. Yago T. McEver R.P. Zhu C. Nature. 2003; 423: 190-193Crossref PubMed Scopus (806) Google Scholar). Fig. 1D exemplifies how the amplitude of force fluctuations in Fig. 1C was reduced by a sliding average of 10 points, such that the levels of force-scan traces before and after the rupture event no longer overlap. This is equivalent to trading the temporal resolution (reduced from 0.2 to 2 ms) for the force resolution. Analysis of Lifetimes—Lifetimes were analyzed using the first-order dissociation kinetics model, Bkoff→R+L where R, L, and B denote receptor, ligand, and bond, respectively. For lifetimes measured at constant force f, koff is assumed to be a constant at each force and independent of time t. This model predicts that lifetimes of single bonds are exponentially distributed, p = exp(-koff(f)t), where p is the probability of a bond observed at time 0 that remained intact at time t. Taking the semi-log linearizes the exponential distribution; thus the slope of the ln(number of events with a lifetime >t) versus t plot equals -koff. It should be noted that the scattering of the individual lifetime data is not a reflection of the lack of measurement accuracy, which is better than 2 ms for the AFM experiments and 4 ms for the flow chamber experiments with the high speed camera. Rather, it is a manifestation of individual molecular bond dissociation, which is stochastic by nature. Thus, the S.D. σ(t) should not be used as error bars because it measures a statistical property of the probability distribution rather than the uncertainty in the data. Indeed, it can be easily shown that σ(t) of an exponential distribution is equal to the mean 〈t〉, both of which equal the reciprocal of off-rate, 1/koff. Several methods were employed to assess the statistical significance of the differences in the koff values estimated at different forces, especially in the force range where catch bonds were observed. First, individual AFM experiments were performed to measure a few lifetimes at each of several force levels that covered the entire force range. The off-rate at each force level was estimated from the reciprocal of the mean and of the S.D. of these measurements. It was found that the koff versus f curves so obtained were stable as soon as the number of measurements reached ∼50. To ensure statistical stability, the data shown in Fig. 4 include 350-400 measurements for L-selectin interacting with either form of PSGL-1 and with endoglycan. The larger data set allowed us to examine the distributions of lifetimes using the ln(number of events with a lifetime ≥ t) versus t plots (Fig. 3), which used the negative slope of the linear fit as estimates for koff. The 95% confidence intervals of the slopes were used to measure the uncertainties of the koff values (shown as error bars in Fig. 4). The statistical significance of differences between any two neighboring points in the catch bond range (where the majority of the data reside) was determined by the F test that compared the slopes of the two ln(number of events with a lifetime ≥ t) versus t data. Coupling L-selectin-Ig to Microspheres and sPSGL-1 and DREG56 to Flow Chamber—1 × 108 polystyrene microspheres (6 μm diameter; Polysciences, Inc.) were incubated with 200 μg/ml anti-human IgG Fc antibody in 500 μl of HBSS overnight at 4 °C. Microspheres were washed three times with HBSS and incubated with 1% human serum albumin (HSA) in HBSS for 2 h at room temperature to block nonspecific binding. After washing with HBSS, anti-human IgG antibody-coated microspheres were incubated with L-selectin-Ig for 1 h at 4 °C in 500 μl of HBSS. A 30-μl drop of streptavidin (50 μg/ml; Pierce) or DREG56 (0.3 μg/ml) was placed in a demarcated area on a 35-mm tissue culture plate (Corning Glass) and incubated at 4 °C overnight. The area was washed twice with HBSS and then blocked with HBSS containing 1% HSA at room temperature for 2 h. The streptavidin-coated plates were further functionalized by incubation with biotinylated sPSGL-1 at 4 °C for 1 h. Transient Tether Lifetime Measurements—Transient tether lifetimes as a function of wall shear stress were measured at low densities of sPSGL-1 or of DREG56 using a flow chamber as described previously (18.Yago T. Leppänen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). Images of transient tethers were captured with a FASTCAM-Super 10 K high speed digital video camera (Photron, San Jose, CA) at 250 frames/s. Images were played back in slow motion, and the durations of transient tethers were measured by using frame-by-frame analysis with a previously described imaging system (18.Yago T. Leppänen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar). Four sets of lifetimes (∼100 tethering events in each set) were measured for each interaction at each wall shear stress. Each set was analyzed by the ln(number of events with a lifetime ≥ t) versus t plot, which was fitted by a straight line. The correlation coefficients, R2, were >0.9 for all fits. Means and standard deviations of lifetimes at each wall shear stress level were also calculated. Off-rates were derived from tether lifetimes by the same methods used for the AFM experiments. The mean ± S.D. of the negative slope, 1/〈t〉, and 1/σ(t) of four independent sets of experiments were plotted against the wall shear stress (Fig. 5). The statistical significance of differences at any two neighboring wall shear stresses was assessed by the Student's t test. Tether Lever Arm Measurements—The force on the tether of a microsphere was calculated based on the tether angle, which was derived from the tether lever arm, i.e. the lateral distance on the flow chamber floor between the contact point of the microsphere and the anchor point of sPSGL-1 or DREG56. The lever arm of the L-selectin-DREG56 tether, which was directly measured by a previously described flow reversal method (18.Yago T. Leppänen A. Qiu H. Marcus W.D. Nollert M.U. Zhu C. Cummings R.D. McEver R.P. J. Cell Biol. 2002; 158: 787-799Crossref PubMed Scopus (140) Google Scholar), was nearly constant (0.96 ± 0.30 μm) across a range of wall shear stresses (0.25-1.6 dynes/cm2). The flow reversal method could not measure the lever arm of the L-selectin-sPSGL-1 tether due to its much faster dissociation. We theref