Csilla Csanyi1, Timea Feller1, Miklos Kellermayer1, Jolan Harsfalvi1
1Department of Biophysics and Radiation Biology, Semmelweis University, Budapest
The largest multimer glycoprotein in plasma is von Willebrand factor (VWF). The globular VWF at the site of injury elongates at high shear rate and cryptic binding sites become accessible for the positive sites of subendothelial collagen and the negative sites of platelet GPIbα receptor, thus VWF mediates platelet adhesion. In pathologic states VWF mediates platelet adhesion to the intact endothelium, with binding to heparan sulphate.
Our aim was to examine with atomic force microscope (AFM) whether the morphology of VW multimers bound to surfaces with different electrostatic characteristics is different.
Solution of heparin affinity chromatography isolated VWF -10ul, 1ng/ul in PBS pH7.4 or 6.0- was dropped on negatively charged mica or on positively charged poly-L-lysin (PLL) treated mica discs; after 1min washed with water (18MΩ*cm), and dried with N2; then analysed with AFM in AC air topography mode.
VWF showed bead-conglomerate structure on PLL and beads-on-string structure on mica. I quantified the shape of particles (n~1000) with circularity what is the ratio of the area of the particle divided by the area of a circle with an equivalent perimeter length. The more circular a particle the closer to 1 this value will be. Our medians (IQR) of circularity are the following: 0.48 (0.33-0.67) on mica; 0.60 (0.43-0.76) on PLL at pH7.4 and 0.51 (0.36-0.67) on mica; 0.66 (0.50-0.79) on PLL at pH6.0.
On AFM images the VW multimers look more like beads-on-string on mica, however they form more circular structures on PLL surface at pH7.4. It is known that the VW multimers have compact structure in Golgi (pH6.0). VWF from pH6.0 buffer on mica have similar structure, like from pH7.4, however, on PLL it is more circular. The negatively charged mica and pH7.4 is better to further examine the topography of VWF.
Doctoral School: Basic and Translational Medicine
Program: Cellular and Molecular Biophysics
Supervisor: Jolan Harsfalvi
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