Solution Structure of Human von Willebrand Factor Studied Using Small Angle Neutron Scattering

Abstract

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...

Protein mobilities and P-selectin storage in Weibel–Palade bodies

“…Strong interactions of this protein with endogenous ProVWF seem unlikely because eotaxin-3 does not associate with VWF or Proregion under neutral conditions (pH 7.4) (Babich et al, 2009), similar to those that exist in the HUVEC ER (pH 7.2) (Erent et al, 2007). Large differences in apparent D of ProVWF-EGFP relative to ssEGFP alone (~50-fold) is unlikely to be due entirely to their different molecular masses (the predicted ratio of D assuming globular shapes is ~3), but rather to other factors such as the highly prolate molecular shape of soluble ProVWF (Singh et al, 2006) or interactions of ProVWF-EGFP with ER-resident components, possibly through its extensive sugar modifications. In common with the size dependences of mobilities in other cellular compartments (Dross et al, 2009;Lukacs et al, 2000;Papadopoulos et al, 2000;Seksek et al, 1997), the diffusion of soluble secretory proteins within the ER of HUVECs slowed sharply with increased protein size: the dependence of D on approximate molecular mass had a -1.6 logarithmic slope instead of the -0.33 slope predicted by the Stokes-Einstein formula (Fig.…”

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

“…For this purpose, we employed a water-soluble, non-cleavable, homobifunctional amine-reactive cross-linker BS3 with a history of successful use in stabilizing protein structures [10][11][12][13] . Figure 5 shows the chemical formula of BS3 and its cross-linking reaction with free epsilon amino groups of lysine in Strep-Tactin.…”

Kinetic study of von Willebrand factor self-aggregation induced by ristocetin

“…Notably, that VWF could self associate and aggregate under high shear stress was known from previous studies. For instance, a shear-dependent self-association of VWF in suspension was demonstrated in a cone-plate viscometer [5]. These studies showed that in all cases the aggregation and web-formation process is essentially linked to the mechanical force generated by shear.…”

“…At sites of high blood flow, von Willebrand factor (VWF) plays a central role in this process [1,2], as it primarily modulates platelet-subendothelium interaction by linking the platelet receptor GpIb/IX/V to the extracellular matrix [3,4]. In fact, when immobilized on subendothelial matrix, VWF forms a reactive surface capable of capturing platelets from flowing blood [5,6]. High shear stress causes micro-and macro-conformational changes of VWF from a globular state to a stretched chain [7].…”

“…This process consists of a conformational change of VWF molecules, that expose within the A1 domain the binding site for GpIbα. This mechanism is triggered either by mechanical forces, such as high shear rate (>5000 sec -1 ) found in the arterial circulation, or by chemical potential generated by interaction with external surfaces and chemicals/snake venoms [5,[11][12][13][14][15]. Finally, natural mutations causing type 2B von Willebrand disease (VWD 2B) such as the Arg1306Trp or Arg1341Trp [6], are also able to stabilize a…”