Shear-dependent morphology of von Willebrand factor bound to immobilized collagen

• Published in: Blood 2002-03, Vol.99 (6), p.2070-2076
• Main Authors: Novák, Levente, Deckmyn, Hans, Damjanovich, Sándor, Hársfalvi, Jolán
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood coagulation. Blood cells; Blood Platelets - metabolism; Cattle; Collagen Type I - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Immunohistochemistry - methods; Microscopy, Atomic Force - methods; Molecular and cellular biology; Perfusion; Platelet; Platelet Adhesiveness; Protein Binding; Stress, Mechanical; von Willebrand Factor - chemistry; von Willebrand Factor - metabolism; von Willebrand Factor - ultrastructure
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V99.6.2070

We have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration–purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m2 = 0.7 dynes/cm2) and high (4.55 N/m2 = 45.5 dynes/cm2) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF “uncoiling” either at a high or at a low shear regime. After perfusing 1 µg/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 × 109/L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for glycoprotein Ib.