Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity

Abstract

Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.

Bulk and Micropatterned Conjugation of Extracellular Matrix Proteins to Characterized Polyacrylamide Substrates for Cell Mechanotransduction Assays

“…Because glycosylated proteins, such as collagen and other ECM proteins, are easily modified to contain aldehyde groups by oxidation with sodium m -periodate (14), this method offers a very efficient and simple conjugation method to polyacrylamide gels (Figure 2A, bottom). In addition to glycosylated proteins, this method will also work with proteins containing a primary amine and a secondary hydroxyl on adjacent carbon atoms as is the case with hydroxylysine, which is prevalent in collagen, and N-terminal serine and threonine residues (15).…”

Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility

“…The cellular response induced by collagen is mainly mediated through the amino acid sequence arginine-glycine-asparagine (RGD) which is recognized by the integrin receptors located at the cell membrane [9,10]. For this reason, collagen has successfully been used to modify biomaterial surfaces in order to enhance their bioactivity, which has been shown, for example, on surfaces of polyurethane [11], polystyrene [12], titanium [13] and titanium alloy [14].…”

The von Willebrand factor collagen-binding activity assay: clinical application

“…The different polystyrene microplates had different effects on the collagen-VWF interaction: some microplates caused a lower number of collagen-binding sites to be available for interaction with the VWF molecule. Previous studies have used the covalent linkage of collagen onto the surface, which overcomes the problem of steric hindrance of the large VWF molecule [9,27]. We had good results in the CBA assay with the Titertek non-activated lowcost immunoplate.…”

“…Surface plasmon technology shows excellent binding of collagen type III to VWF [27]. Because of these advantages, collagen type III has been widely used in CBA assays [7,9,27]. An optimal collagen concentration (10 µg/ml) provided a low variation coefficient (4.8% for normal values) and good discrimination between different types of VWD.…”