von Willebrand factor (VWF) binding to platelets under high fluid shear is an important step regulating atherothrombosis. We applied light and small angle neutron scattering to study the solution structure of human VWF multimers and protomer. Results suggest that these proteins resemble prolate ellipsoids with radius of gyration (R g ) of ϳ75 and ϳ30 nm for multimer and protomer, respectively. The ellipsoid dimensions/radii are 175 ؋ 28 nm for multimers and 70 ؋ 9.1 nm for protomers. Substructural repeat domains are evident within multimeric VWF that are indicative of elements of the protomer quarternary structure (16 nm) and individual functional domains (4.5 nm). Amino acids occupy only ϳ2% of the multimer and protomer volume, compared with 98% for serum albumin and 35% for fibrinogen. VWF treatment with guanidine⅐HCl, which increases VWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales <10 nm without altering protein R g . Treatment of multimer but not protomer VWF with random homobifunctional linker BS 3 prior to reduction of intermonomer disulfide linkages and Western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable intermonomer non-covalent interactions within the multimer. Overall, multimeric VWF appears to be a loosely packed ellipsoidal protein with non-covalent interactions between different monomer units stabilizing its solution structure. Local, and not large scale, changes in multimer conformation are sufficient for ADAMTS-13-mediated proteolysis.von Willebrand factor (VWF) 2 is a large, multidomain glycoprotein that is present in human blood and in secretory granules of endothelial cells and platelets (1-3). This protein occurs both as a protomer and in multimeric form. The ϳ500-kDa protomer consists of two identical monomer subunits linked at the C terminus by disulfide bonds. Linear multimers formed by cysteine-cysteine linkages near the N terminus result in a molecular mass of Ͼ10,000 kDa.VWF serves many functions. The binding of surface-immobilized VWF to platelet receptor GpIb␣ results in intermolecular bonds with high tensile strength (4, 5). This molecular interaction allows platelet capture at sites of vascular injury under high fluid shear conditions. The binding of plasma VWF to platelet receptor GpIb␣ under high hydrodynamic shear also leads to platelet activation and subsequent platelet arrest (6). Various mutations in VWF result in the bleeding defects that characterize von Willebrand disease (1). In blood, VWF binding to pro-coagulation factor VIII increases factor VIII lifetime in circulation. Finally, the size of VWF and its response to fluid flow are key determinants in regulating protein function under physiological and pathological conditions. In support of this, the life threatening systemic illness thrombotic thrombocytopenic purpura (TTP) is attributed to the presence of very large VWF multimers, which are caused by the malfunction or absence of a metalloprotease termed ADAMTS-13 ("a disintegr...

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.

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Soluble oligosaccharides and glycoproteins can inhibit leukocyte adhesion during a range of vascular ailments including inflammation, thrombosis, and cancer metastasis. The design of such molecules in many cases is based on the structure of naturally occurring selectin ligands. In this case, synthetic selectin-ligand mimetics act as competitive inhibitors of cell adhesion. In an alternate approach, cell-permeable, small-molecule oligosaccharides have been shown to alter the metabolic pathways that lead to the biosynthesis of functional selectin-ligands. The addition of such molecules results in glycoproteins that are defective in their ability to bind selectins. Quantitative in vitro testing of the efficacy of the above inhibition strategies ideally requires the application of assays that mimic the in vivo physiological milieu in terms of the valency of selectin and selectin-ligands, the physiological fluid-flow conditions, and the use of blood cells. Assays that are performed in small volumes are preferable when the quantity of available inhibitor is scarce. Finally, the measurements must account for the rapid on- and off-rates of selectin-mediated binding interactions. This chapter addresses these issues by presenting methods to measure selectin function in enzyme-linked immunosorbent assay and flow cytometry-based static assays, cell-adhesion assays performed under shear flow in cone-plate viscometers, and Biacore surface plasmon resonance measurements of molecular-binding kinetics. Examples are presented where such methods are applied to measure the ability of simple oligosaccharides based on sialyl Lewis-X and complex molecules with the core-2 structure to block selectin function. Such methods may be extended to identify potent selectin antagonists in a library of carbohydrates.

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