COLLAGEN COVALENTLY IMMOBILIZED ONTO PLASTIC SURFACES SIMPLIFIES MEASUREMENT OF VON-WILLEBRAND FACTOR-COLLAGEN BINDING-ACTIVITY

Human collagen type III was immobilized covalently via activated carbo hydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activi ty (vWF:CBA) in an ELISA. Such plates were simple to prepare and remai ned stable at 4 degrees C and -20 degrees C for at least 2 months. Sam ples analyzed by this system included (a) normal human vWF fractionate d 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 conce ntration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, w hile higher values were obtained for samples containing these multimer s. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional an d structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vW F which interacts with the glycoprotein Ib completely inhibited the vW F ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. T hus vWF:CBA and vWF:RistCof clearly represent separate, noninterchange able functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen on to microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, th is method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.