Von Willebrand protein binds to extracellular matrices independently of collagen.

Abstract

Platelets contain receptors for vW protein, and monoclonal antibodies against these membrane glycoproteins inhibit the binding of vW protein (9-13). The binding site in the subendothelium has been considered to be collagen, based on vW protein affinity experiments (14, 15) and electron microscopic observations of its proximity to collagen fibers in the subendothelium (16). We have studied the relationship of vW protein and collagen in the extracellular matrix of endothelial cells and of other cells to which vW protein has been added and have found that the presence of vW protein in the matrix is independent of the presence of collagen. Our results strongly indicate that the major binding site for vW protein in the subendothelium is not collagen. MATERIALS AND METHODSCells and Culture Conditions. Endothelial cells were obtained from human umbilical vein by mild proteolytic digestion as described (8). Cells were cultured in McCoy's 5A medium (Flow Laboratories) containing 20% fetal bovine serum. Human foreskin fibroblasts in 16th passage, a gift from M. Menegus (University of Rochester, Rochester, NY), were grown in the same medium. NIL 8 hamster cells were a gift from R. 0. Hynes (Massachusetts Institute of Technology, Cambridge, MA) and were cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 5% fetal bovine serum.Antisera. The preparation and characterization of antisera against human vW protein have been described (8,17). Other antisera were anti-type IV collagen (18), a gift from G. R. Martin (National Institutes of Health, Bethesda, MD), antitype V collagen (19), a gift from H. E. Sage (University of Washington, Seattle, WA), and rhodamine-conjugated antifibronectin (20), supplied by R. 0. Hynes.Immunofluorescence. Cells grown on glass coverslips were fixed in 3.7% HCHO in phosphate-buffered saline (saline) for 20 min and, if desired, permeabilized in 0.5% Triton X-100 in saline for 15 min at room temperature. Incubations with the first antibody and with goat fluorescein isothiocyanate antirabbit immunoglobulin (Miles) were for 30 min at 37°C. AntivW protein antiserum was used at 1:40 dilution; anti-type IV collagen, at 1:10 dilution; and anti-type V collagen, at 1:20 dilution. For double-label fluorescence, the protocol of Hynes and Destree was used (20). After staining with the first antibody and fluorescein-conjugated goat anti-rabbit immunoglobulin, cells were incubated for 30 min with normal rabbit serum (1:10 dilution) and then stained with rhodamine-conjugated anti-fibronectin for 30 min at 37°C. All intermediate washes were in saline. Coverslips were mounted in Gelvatol. The fluorescein and rhodamine stains showed no interference. For example, when anti-vimentin was used as first antibody, the visualized intermediate filament pattern did not coincide with the straighter extracellular fibronectin pattern.Binding of Exogenous vW Protein to Matrices. Cells were either plated in the presence of vW protein-containing media or switched to this medium after they became established. The ...

Role of factor VIII-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III.

“…The absence of a preferred order in the adsorption to collagen makes it unlikely that FVIII-VWF binds to collagen via fibronectin, or the reverse. Recent data from endothelial cell culture studies have suggested that a direct interaction occurs between FVIII-VWF and fibronectin because these proteins codistributed in the subendothelial matrix (50). Our data do not rule out such interaction, but they indicate that both FVIII-VWF and fibronectin from plasma interact directly with collagen and are then able to support platelet adhesion.…”

Evidence that the primary binding site of von Willebrand factor that mediates platelet adhesion on subendothelium is not collagen.

“…On the basis of the results presented in this paper and on the basis of previous fluorescence studies (8,15,16), it can be concluded that collagen is not the primary binding site of von Willebrand factor in the vessel wall in the described system.…”

“…Several groups of investigators have demonstrated that von Willebrand factor binds to collagen types I and III (9)(10)(11)(12)(13)(14), and exposed collagen at the site of vessel injury has been considered to be involved in the binding ofvon Willebrand factor. Recently, Hormia et al and Wagner et al (15,16) have suggested that not collagen but fibronectin may be the binding site of von Willebrand factor.…”

150-kD von Willebrand factor binding protein extracted from human vascular subendothelium is type VI collagen.

“…Firstly, vascular subendothelium lacks significant quantities ofthese types of collagen (13)(14)(15). Also, Wagner et al (16) concluded that the subendothelial binding site is not fibrillar collagen, because they found that collagenase digestion oftissue culture-derived subendothelial matrix did not remove vWF. Also, subendothe-1.…”