INTRODUCTION: Von Willebrand factor (VWF) and platelet binding needs a uniform collagen matrix therefore we aimed to find an optimal condition for the preparation of human type-I and type-III collagen matrices. METHOD: The effects of pH, salt and ligand concentration and binding time were tested when collagen matrices were prepared by adsorption. Surface-bound collagen and collagen-bound VWF measured by specific antibodies. Platelet adhesion was tested under flow conditions at a shear rate of 1800s(-1) for 2 min. Matrices and platelets were visualized by atomic force and scanning electron microscope. RESULTS: The extent of human collagens type-I and III binding to the surface was 10 and 4 times greater and binding was maximal under 8-16 hours, when coated from physiological buffer solution versus acid solution. Collagen fibrils were more developed and platelet adhesion was higher, with more organized and denser aggregates. VWF binding was parallel to the surface bound collagen in both collagen types. CONCLUSION: Collagen coating of surfaces for VWF binding and platelet adhesion studies is very variable from acid solution. Our experiments provide evidences that neutralizing the acid and adding NaCl in physiological concentration, thereby facilitating formation of collagen fibril molecules in solution, results in efficient coating of human type-I and type III collagens, which then bind normal VWF equally well.
INTRODUCTION:Von Willebrand factor (VWF) and platelet binding needs a uniform collagen matrix therefore we aimed to find an optimal condition for the preparation of human type-I and type-III collagen matrices. METHOD: The effects of pH, salt and ligand concentration and binding time were tested when collagen matrices were prepared by adsorption. Surface-bound collagen and collagen-bound VWF measured by specific antibodies. Platelet adhesion was tested under flow conditions at a shear rate of 1800s(-1) for 2 min. Matrices and platelets were visualized by atomic force and scanning electron microscope. RESULTS: The extent of human collagens type-I and III binding to the surface was 10 and 4 times greater and binding was maximal under 8-16 hours, when coated from physiological buffer solution versus acid solution. Collagen fibrils were more developed and platelet adhesion was higher, with more organized and denser aggregates. VWF binding was parallel to the surface bound collagen in both collagen types. CONCLUSION: Collagen coating of surfaces for VWF binding and platelet adhesion studies is very variable from acid solution. Our experiments provide evidences that neutralizing the acid and adding NaCl in physiological concentration, thereby facilitating formation of collagen fibril molecules in solution, results in efficient coating of human type-I and type III collagens, which then bind normal VWF equally well.
Authors: V H Flood; J C Gill; P A Christopherson; J S Wren; K D Friedman; S L Haberichter; R G Hoffmann; R R Montgomery Journal: J Thromb Haemost Date: 2012-07 Impact factor: 5.824
Authors: Veronica H Flood; Joan Cox Gill; Kenneth D Friedman; Pamela A Christopherson; Paula M Jacobi; Raymond G Hoffmann; Robert R Montgomery; Sandra L Haberichter Journal: Clin Chem Date: 2013-01-22 Impact factor: 8.327