Acknowledgements We thank M. Inouye for the gift of the RU1012 strain, L. Loew for the gift of styryl dyes, S. Conrad and G. Shirman for assistance with mutagenesis and protein chemistry, and M. G. Prisant for construction of the computer cluster. This work was supported by grants from the Office of Naval Research, the Defense Advanced Research Project Agency and the National Institutes of Health.Competing interests statement The authors declare that they have no competing financial interests.Correspondence and requests for material should be addressed to H. Bonds between adhesion molecules are often mechanically stressed. A striking example is the tensile force applied to selectin-ligand bonds, which mediate the tethering and rolling of flowing leukocytes on vascular surfaces 1-3 . It has been suggested that force could either shorten bond lifetimes, because work done by the force could lower the energy barrier between the bound and free states 4 ('slip'), or prolong bond lifetimes by deforming the molecules such that they lock more tightly 5,6 ('catch'). Whereas slip bonds have been widely observed 7-14 , catch bonds have not been demonstrated experimentally. Here, using atomic force microscopy and flow-chamber experiments, we show that increasing force first prolonged and then shortened the lifetimes of P-selectin complexes with P-selectin glycoprotein ligand-1, revealing both catch and slip bond behaviour. Transitions between catch and slip bonds might explain why leukocyte rolling on selectins first increases and then decreases as wall shear stress increases 9,15,16 . This dual response to force provides a mechanism for regulating cell adhesion under conditions of variable mechanical stress.Using atomic force microscopy (AFM) (Fig. 1a), we measured the force dependence of bond lifetimes of P-selectin with two forms of P-selectin glycoprotein ligand-1 (PSGL-1) or with G1, a blocking monoclonal antibody (mAb) against P-selectin 17 (see Methods). P-selectin is an extended C-type lectin expressed on activated endothelial cells and platelets. PSGL-1 is a mucin expressed on leukocytes. Ca 2þ -dependent interactions of P-selectin with PSGL-1 mediate the tethering and rolling of flowing leukocytes on vascular surfaces in response to infection or tissue injury 1-3 .We captured dimeric PSGL-1 purified from human neutrophils 18 or monomeric recombinant soluble PSGL-1 (sPSGL-1) 19 with PL2, a non-blocking anti-PSGL-1 mAb 20 adsorbed on the cantilever tip (Fig. 1b). Cantilever tips bearing (s)PSGL-1 or G1 were repeatedly brought into contact with lipid bilayers reconstituted with P-selectin purified from human platelets 21 to allow bond formation. The cantilever was then retracted a prescribed distance to apply a constant tensile force to the bond or bonds (if any resulted from the contact), and the duration or lifetime of the adhesion at that force was recorded (Fig. 1c). To measure lifetime at forces lower than the level of their fluctuations, many instantaneous forces were averaged (Fig. 1d, e). This enabled the reli...

Abstract. Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize proteindependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of '25I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectinexpressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses. TH~ selectins are a family of three Ca2+-dependent membrane-bound lectins that initiate the rolling adhesion of leukocytes to platelets or endothelial cells under the shear forces found in the venular circulation (6,25,33). L-selectin, expressed on leukocytes, binds to constitutive or inducible ligands on endothelial cells. E-selectin, expressed by cytokine-activated endothelial cells, and P-selectin, expressed by thrombin-activated platelets and endothelial cells, bind to ligands on myeloid cells and subsets of lymphocytes. Although the selectins interact weakly with small sialylated, fucosylated oligosaccharides such as sialyl Lewis x (Galfll-4[Fuccd-3lGlcNAc) (Le~) ~ (17, 50), they bind with higher affinity to glycans displayed on a limited number of glycoproteins (3,5,20,26,28,29,39,52) or proteoglycans (41). A major unresolved issue is whether selectins must bind to these higher affinity, but less abundant, ligands in order for leukocytes to roll on the vessel wall un-

Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds.

Human blood monocytes adhere rapidly and for prolonged periods to activated platelets that display P-selectin, an adhesion protein that recognizes a specific ligand on leukocytes, P-selectin glycoprotein-1. We previously demonstrated that P-selectin regulates expression and secretion of cytokines by stimulated monocytes when it is presented in a purified, immobilized form or by transfected cells. Here we show that thrombin-activated platelets induce the expression and secretion of monocyte chemotactic protein-1 and IL-8 by monocytes. Enhanced monokine synthesis requires engagement of P-selectin glycoprotein-1 on the leukocyte by P-selectin on the platelet. Secretion of the chemokines is not, however, directly signaled by P-selectin; instead, tether-

We used an immunoperoxidase procedure to examine the tissue distribution of the platelet a-granule membrane protein, GMP-140. In addition to its presence in megakaryocytes and platelets, GMP-140 antigen was found in vascular endothelial cells of diverse human organs, but it was not detected in other types of secretory cells. 35S]Cysteine-labeled human umbilical vein endothelial cells synthesized a GMP-140 molecule containing complex N-linked oligosaccharides similar to those previously demonstrated in platelets and the megakaryocytic HEL cell line. Using an immunogold procedure on frozen thin sections of endothelial cells, we found GMP-140 antigen to be localized to membranes of electron-dense storage granules. In double-label experiments there was colocalization of GMP-140 with vWf, indicating that these granules are Weibel-Palade bodies. When endothelial cells were stimulated with histamine, GMP-140 rapidly redistributed to the plasma membrane. Immunoassays of cell lysates indicated that, relative to total cell protein, less GMP-140 is present in human umbilical vein endothelial cells than in platelets. The restricted expression of GMP-140 in secretory granules of platelets and endothelium suggests that it has a specific function in the vascular system rather than a general role related to inducible secretion.

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Granule membrane protein-140 (GMP-140), a membrane glycoprotein of platelet and endothelial cell secretory granules, is rapidly redistributed to the plasma membrane during cellular activation and degranulation. Also known as PADGEM protein, GMP-140 is structurally related to two molecules involved in leukocyte adhesion to vascular endothelium: ELAM-1, a cytokine-inducible endothelial cell receptor for neutrophils, and the MEL-14 lymphocyte homing receptor. These three proteins define a new gene family, termed selectins, each of which contains an N-terminal lectin domain, followed by an epidermal growth factor-like module, a variable number of repeating units related to those in complement-binding proteins, a transmembrane domain, and a short cytoplasmic tail. Here we demonstrate that GMP-140 can mediate leukocyte adhesion, thus establishing a functional similarity with the other selectins. Human neutrophils and promyelocytic HL-60 cells bind specifically to COS cells transfected with GMP-140 complementary DNA and to microtitre wells coated with purified GMP-140. Cell binding does not require active neutrophil metabolism but is dependent on extracellular Ca2+. Within minutes after stimulation with phorbol esters or histamine, human endothelial cells become adhesive for neutrophils; this interaction is inhibited by antibodies to GMP-140. Thus, GMP-140 expressed by activated endothelium might promote rapid neutrophil targeting to sites of acute inflammation.

Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged ("catch") and then shortened ("slip") bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flowenhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif-13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.