von Willebrand Factor (vWF) plays central role in regulation of hemostasis and thrombosis by participating in blood coagulation and facilitating adhesion and aggregation of platelets. The dual roles are integral to the maintenance of hemostasis after vasculature injury or in the presence of an implanted cardiovascular device. While the general sequence of functional events involving vWF is known, the corresponding structural changes in vWF have not been determined on a molecular scale under aqueous conditions. The proposed studies focus on elucidation of the three (3D) dimensional (tertiary) structure of vWF under physiologic and pathophysiologic shear conditions using the novel technique of atomic force microscopY (AFM). The central hypothesis to be examined is that there is a close relationship between the conformational state of vWF and expression of the protein's functional binding domains.
The specific aims of this project include: (1) determination of the 3D conformation of vWF multimers on different adhesive substrata (collagens, heparin, artificial surfaces) under hemodynamic conditions of increasing shear stress; (2) identification of the Factor VIII functional binding domain in the tertiary structure of surface adsorbed vWF; (3) identification and mapping on a sub-nanometer scale of exposed surface charge domains of vWF; and (4) quantification of the intermolecular adhesive forces involved in specific vWF ligand-receptor interactions including peptide segments of the Al domain and RGDX ligand binding to platelet integral membrane receptors GPIb and GPIIb-IIIa and to collagen or heparinized substrata. The unique capabilities of AFM to measure both 3D structure and intermolecular forces will be the central technique used in these studies. A range of in vitro dynamic flow conditions extending from stasis to pathophysiologic shear stresses will be quantified and controlled using a rotating disk system and a laminar flow chamber. Conformational studies will be directly correlated to the functional state of vWF using complimentary assays including platelet adhesion determined by high resolution fluorescence microscopy, and by FITC labeled and gold bead labeled monoclonal antibodies to known vWF epitopes. These studies will contribute new insights and understanding of vWF structure-function relations under a range of shear conditions and in developing a comprehensive understanding of thrombogenesis.
