Critical Extracellular Matrix Proteins Besides Collagen Bind Von Willebrand Factor

Introduction: Vascular endothelial injury exposes blood to many extracellular matrix proteins including collagen. Von Willebrand Factor (VWF) is a coagulation factor that binds platelets to collagen in the process of normal hemostasis. Collagen IV has been shown to bind VWF through the A1 domain. Many adhesive glycoproteins found in the extracellular matrix of vascular endothelium have been shown to induce hemostasis by binding coagulation factors. We hypothesized that in addition to collagen, other extracellular matrix proteins can bind VWF as well. We investigated VWF's interactions with four other extracellular matrix (ECM) proteins: laminin, fibronectin, thrombospondin, and vitronectin.

Methods: Enzyme linked immunoassay (ELISA) was performed to measure the ability of laminin, fibronectin, thrombospondin, and vitronectin to bind to VWF. Each ECM protein was coated individually on a maleic anhydride plate. Detection of VWF:ECM complexes was performed using a combination of two biotinylated monoclonal anti-VWF antibodies. Inhibition of VWF:ECM interactions was tested using monoclonal and polyclonal antibodies against VWF and the individual ECM proteins. We also tested inhibition of VWF:ECM interactions by competing with collagen IV, laminin, fibronectin, thrombospondin, and vitronectin. ELISA was also used to test laminin, fibronectin, thrombospondin, and vitronectin binding to recombinant VWF (rVWF) constructs to determine a specific binding region and to determine the requirement for high molecular weight multimers with each ECM protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on all ECM proteins to investigate purity.

Results: Results for the four ECM proteins generally paralleled each other and will be referred to together unless otherwise specified. SDS-PAGE, however, showed independent bands for each ECM protein preparation, confirming that these interactions were with the individual proteins and not with a single protein contaminating all preparations. ELISA showed binding of laminin, fibronectin, thrombospondin, and vitronectin to wild type (WT) rVWF and plasma from heathy individuals with normal VWF. No binding was detected for individuals with type 3 VWD. Polyclonal anti-VWF (DAKO) inhibited all VWF:ECM interactions (average 99% inhibition), indicating that binding is VWF specific. Monoclonal anti-VWF A1 domain (AVW-3) also inhibited all VWF:ECM interactions (average 98% inhibition), confirming that the A1 domain of VWF is crucial for this interaction. Several rVWF variants were used to test the importance of VWF functional domains. Compared to WT rVWF, VWF:ECM binding was reduced or undetectable with all ECM proteins for 1395A (undetectable) and 1399H (average 4%) but normal for 1786D, 1392A and 1402A, which were shown to abrogate VWF binding to collagen III and collagen IV, respectively. An 11 amino acid deletion from 1392 to 1402 also showed undetectable binding. Reduced binding with these constructs indicate the critical nature of the 1395 to 1399 amino acid region of the A1 domain to the VWF:ECM interactions. Compared to WT rVWF, binding was decreased or undetectable on all ECM proteins for 1597W (average 26%), 2773R (undetectable) and Y87S (undetectable) rVWF constructs. The 1597W, 2773R, and Y87S rVWF constructs contain defects in domains of VWF crucial to multimerization. Reduced binding with these constructs indicate that the VWF:ECM interactions are multimer dependent. All VWF:ECM interactions were significantly inhibited by collagen IV (90% inhibition for thrombospondin, complete inhibition for all other ECMs). The VWF:ECM interactions were also partially inhibited by thrombospondin (average 46% inhibition) and vitronectin (average 62% inhibition). Laminin and fibronectin did not noticeably inhibit any VWF:ECM interactions.

Conclusions: In addition to collagen, VWF binds other ECM proteins found in vascular endothelium including laminin, fibronectin, thrombospondin, and vitronectin. These four ECM proteins appear to bind to VWF in a restricted region of the A1 domain as competitive binding suggests differences between collagen IV and other ECM proteins. The role and importance of VWF's interaction with these ECM proteins offer alternative binding partners that may represent a unique contribution to hemostasis.