We have previously demonstrated that the isolated von Willebrand Factor (vWF)-Al-domain can activate platelets in a GPIb-dependent manner. Here we evaluated the functional impact of targeted point mutations Gly561His (G1324(561)H), an analog of a previously described 2M von Willebrand disease variant, and Asp560Ser (D1323(560)S) in the model of the membrane expressed A1-domain. Platelet aggregation in response to COS-7-cells stably transfected with wild type A1-domain was abrogated by both substitutions. Ristocetin did not increase the aggregatory potential of mutant vWF-A1, in contrast to native forms. Botrocetin boosted the aggregatory responses of all A1-domains tested. These data suggest that G1324(561) and D1323(560) comprise part of the GPIb binding motif essential for subsequent platelet aggregation. Botrocetin seems to alter the potential of vWF for GPIb interaction independently of that motif. The experimental system tested here provides a rapid and reproducible approach for the functional analysis of isolated A1-domain interactions with platelet-GPIb. (C) 2003 Elsevier Science (USA). All rights reserved.