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...

We tested the possibility that immune complexes formed following platelet factor 4 (PF4/CXCL4) binding to anti-PF4 antibody can stimulate neutrophil activation, similar to previous reports with platelets. Monoclonal Abs against PF4 and IgG from a heparin-induced thrombocytopenia (HIT) patient were applied. We observed that although PF4 or anti-PF4 antibody alone did not alter neutrophil function, costimulation with both reagents IntroductionPlatelet factor 4 (PF4/CXCL4) is an ELR Ϫ tetrameric, cationic chemokine that constitutes 25% of the protein in platelet ␣-granules. 1,2 It is also found bound to the luminal vascular endothelial surface. 3,4 Although platelets represent the primary source of PF4, a recent report 5 also suggests that the protein is expressed at lower levels in other cells of the immune system including cultured T cells, monocytes, and endothelial and smooth muscle cells. Treatment of patients with heparin results in PF4-heparin complex formation, and a dramatic increase in blood concentration of PF4. Heparin-PF4 binding is facilitated by the multivalent nature of both heparin and PF4. Whereas PF4 binds heparin with relatively high apparent affinity (K d , ϳ 4-20 nM), 6 binding interactions with glycosaminoglycans including those on the neutrophil surface are weaker (K d , ϳ 650 nM). 7 PF4 is typically at less than 1 nM in normal serum, and this level rises to 0.4 to 2.5 M upon platelet activation. 1,2 One to five percent of patients receiving unfractionated heparin suffer from heparin-induced thrombocytopenia (HIT). 8,9 In these cases, PF4-heparin complexes trigger an immune response and the generation of anti-PF4 antibodies. Macromolecular antibodyheparin-PF4 immune complexes then bind Fc␥RIIa (CD32a) on platelets and induce platelet activation. 4,10,11 Using chondroitinase ABC to cleave cell surface glycosaminoglycans, Rauova et al 12 suggest a role for platelet chondroitin sulfates in cell activation. Platelet activation in turn results in more PF4 release into circulation, enhanced immune-complex formation, exaggerated platelet activation, and cell clearance.Ten to fifty percent of HIT patients experience thrombosis in the arterial and venular circulation. This can lead to limb-and life-threatening complications. Although the precise mechanism(s) of thrombosis is yet unestablished, evidence in literature supports a role for activated platelets, resulting procoagulants, and microparticles. 13 Immune complexes also bind to endothelial cells 3,4 and they may up-regulate adhesion molecules that are typically associated with inflammatory ailments including E-/P-selectin, VCAM-1, and ICAM-1. 14 The release of tissue factor and interleukin-8 by monocytes is an additional feature that is thought to contribute to thrombosis. 15,16 Finally, plasma from HIT patients has been shown to activate neutrophils and enhance platelet-neutrophil adhesion via yet unidentified mechanisms. 17 In the current paper, we examined the effect of PF4, heparin, and anti-PF4 antibodies (monoclonals and polyclonal HIT pa...

Platelet-leukocyte adhesion may contribute to thrombosis and inflammation. We examined the heterotypic interaction between unactivated neutrophils and either thrombin receptor activating peptide (TRAP)-stimulated platelets or P-selectin-bearing beads (Ps-beads) in suspension. Cone-plate viscometers were used to apply controlled shear rates from 14 to 3000/s. Platelet-neutrophil and bead-neutrophil adhesion analysis was performed using both flow cytometry and high-speed videomicroscopy. We observed that although blocking antibodies against either P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1) alone inhibited platelet-neutrophil adhesion by approximately 60% at 140/s, these reagents completely blocked adhesion at 3000/s. Anti-Mac-1 alone did not alter platelet-neutrophil adhesion rates at any shear rate, though in synergy with selectin antagonists it abrogated cell binding. Unstimulated neutrophils avidly bound Ps-beads and activated platelets in an integrin-independent manner, suggesting that purely selectin-dependent cell adhesion is possible. In support of this, antagonists against P-selectin or PSGL-1 caused dissociation of previously formed platelet-neutrophil and Ps-bead neutrophil aggregates under shear in a variety of experimental systems, including in assays performed with whole blood. In studies where medium viscosity and shear rate were varied, a shear threshold for P-selectin PSGL-1 binding was also noted at shear rates <100/s when Ps-beads collided with isolated neutrophils. Results are discussed in light of biophysical computations that characterize the collision between unequal-size particles in linear shear flow. Overall, our studies reveal an integrin-independent regime for cell adhesion and weak shear threshold for P-selectin PSGL-1 interactions that may be physiologically relevant.

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