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Title: Solution structure of human von Willebrand factor studied using small angle nuetron 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 (Rg) of ~75nm and ~30nm for multimer and protomer respectively. The ellipsoid dimensions/radii are 175×28nm for multimers and 70×9.1nm for protomers. Substructural repeat domains are evident within multimeric-vWF that are indicative of elements of the protomer quarternary structure (16nm) and individual functional domains (4.5nm). Amino acids occupy only ~2% volume of multimer and protomer ellipsoids, compared to other proteins like albumin (98%) and fibrinogen (35%). vWF treatment with Guanidine·HCl, which increases vWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales<10nm without altering protein Rg. Treatment of multimer but not protomer-vWF with random homobifunctional linker BS3, prior to reduction of inter-monomer disulfide linkages and western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable inter-monomer non-covalent interactions within the multimer. Overall, vWF solution structure is stabilized by non-covalent interactions between different monomer units. Local changes in multimer conformation are sufficient for ADAMTS-13 mediated proteolysis.

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
918859
Report Number(s):
PNNL-SA-49749
Journal ID: ISSN 0021-9258; JBCHA3; TRN: US0805917
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 281; Journal Issue: 50
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS; ALBUMINS; AMINO ACIDS; DIMERS; DISULFIDES; FIBRINOGEN; FUNCTIONALS; GUANIDINES; MONOMERS; NEUTRONS; PROTEINS; PROTEOLYSIS; SCATTERING; SHEAR

Citation Formats

Singh, Indrajeet, Shankaran, Harish, Beauharnois, Mark E, Xiao, Zhihua, Alexandridis, Paschalis, and Neelamegham, Sriram. Solution structure of human von Willebrand factor studied using small angle nuetron scattering. United States: N. p., 2006. Web. doi:10.1074/jbc.M607123200.
Singh, Indrajeet, Shankaran, Harish, Beauharnois, Mark E, Xiao, Zhihua, Alexandridis, Paschalis, & Neelamegham, Sriram. Solution structure of human von Willebrand factor studied using small angle nuetron scattering. United States. doi:10.1074/jbc.M607123200.
Singh, Indrajeet, Shankaran, Harish, Beauharnois, Mark E, Xiao, Zhihua, Alexandridis, Paschalis, and Neelamegham, Sriram. Fri . "Solution structure of human von Willebrand factor studied using small angle nuetron scattering". United States. doi:10.1074/jbc.M607123200.
@article{osti_918859,
title = {Solution structure of human von Willebrand factor studied using small angle nuetron scattering},
author = {Singh, Indrajeet and Shankaran, Harish and Beauharnois, Mark E and Xiao, Zhihua and Alexandridis, Paschalis and Neelamegham, Sriram},
abstractNote = {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 (Rg) of ~75nm and ~30nm for multimer and protomer respectively. The ellipsoid dimensions/radii are 175×28nm for multimers and 70×9.1nm for protomers. Substructural repeat domains are evident within multimeric-vWF that are indicative of elements of the protomer quarternary structure (16nm) and individual functional domains (4.5nm). Amino acids occupy only ~2% volume of multimer and protomer ellipsoids, compared to other proteins like albumin (98%) and fibrinogen (35%). vWF treatment with Guanidine·HCl, which increases vWF susceptibility to proteolysis by ADAMTS-13, causes local structural changes at length scales<10nm without altering protein Rg. Treatment of multimer but not protomer-vWF with random homobifunctional linker BS3, prior to reduction of inter-monomer disulfide linkages and western blotting reveals a pattern of dimer and trimer units that indicate the presence of stable inter-monomer non-covalent interactions within the multimer. Overall, vWF solution structure is stabilized by non-covalent interactions between different monomer units. Local changes in multimer conformation are sufficient for ADAMTS-13 mediated proteolysis.},
doi = {10.1074/jbc.M607123200},
journal = {Journal of Biological Chemistry},
number = 50,
volume = 281,
place = {United States},
year = {Fri Dec 15 00:00:00 EST 2006},
month = {Fri Dec 15 00:00:00 EST 2006}
}
  • von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, can result from either a quantitative or a qualitative defect in the adhesive glycoprotein, von Willebrand factor (vWF). Molecular studies of vWD have been limited by the large size of the vWF gene and difficulty in obtaining the vWF mRNA from patients. By use of an adaptation of the polymerase chain reaction, vWF mRNA was amplified and sequenced from peripheral blood platelets. A silent vWF allele was identified, resulting from a cis defect in vWF mRNA transcription or processing. In two type IIA vWD patients, two different butmore » adjacent missense mutations were identified, the locations of which may identify an important vWF functional domain. Expression in heterologous cells of recombinant vWF containing one of these latter mutations reproduced the characteristic structural abnormality seen in type IIA vWD plasma.« less
  • Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus snake venom, inhibits platelet aggregation and the binding of {sup 125}I-fibrinogen to ADP-stimulated platelets without affecting the platelet-release reaction. {sup 125}I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. {sup 125}I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS). The authors determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acidmore » sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen {alpha}{sub 1} (I) and laminin B{sub 1}, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. In conclusion, the data suggest that the biological activity of trigramin may depend upon the presence of an RGD sequence, the secondary structure of the molecule, and perhaps some other sequences that it shares with adhesive proteins.« less
  • Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 {angstrom}, and its maximum dimension is approximately 275-295 {angstrom}. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.
  • Human plasma fibronectin has been investigated at physiological pH and ionic strength, by using small-angle X-ray and neutron scattering techniques. The results indicate that the molecule is disc shaped with an axial ratio of about 1:10. In fact, an ellipsoid of revolution with semiaxes a = 1.44 nm and b = c = 13.8 nm is in agreement with the experimental scattering data, and can also fully explain the rather extreme hydrodynamic parameters reported for fibronectin. The X-ray data gave a radius of gyration of 8.9 nm and a molecular weight of 510,000, whereas the neutron data gave slightly largermore » values, 9.5 nm and 530,000, respectively. From the volume of the best fitting ellipsoid we obtain a degree of hydration of 0.61 g H/sub 2/O/g protein (dry weight). Neutron data, recorded at different D/sub 2/O concentrations in the solvent, gave a match point of 43% D/sub 2/O, which indicates that approximately 80% of the hydrogens bound to oxygen and nitrogen are exchangeable.« less
  • Washed human platelets in buffers containing either 2 mM Ca++ or 4 mM EDTA were stimulated by human alpha-thrombin to induce secretion. The binding of two endogenous secreted proteins, factor-VIII-related protein (VIII-R) (von Willebrand factor) and platelet factor 4, was measured by reacting thrombin-treated and control platelets with specific antibodies to these proteins, then quantifying antibody binding with /sup 125/I-staphylococcal protein A. Both of these granule proteins were associated with the platelet membrane surface by a calcium-dependent mechanism after thrombin-induced secretion. This ability to bind endogenous secreted proteins to the plasma membrane surface may provide a mechanism by which themore » platelet can concentrate and organize its secreted proteins for subsequent physiologic reactions.« less