Molecular mechanism of VWF self-association. (A) Four VWF mutants lacking the A1 domain were produced, and each was labeled with Alexa 647. (B-E) PRP was diluted 50-fold into HEPES buffer containing one of the ΔA1-Alexa 647 variants (2.5 µg/mL), along with either 5 µg/mL WT-VWF (B-C) or Lock-VWF (D-E). The mixture was sheared at 9600/s using a viscometer in buffer containing either no exogenous calcium (B,D) or physiological calcium (C,E). VWF self-association was measured based on ΔA1-Alexa 647 binding to platelets using flow cytometry. VWF self-association was observed even when the ΔA1-proteins were sheared in the presence of Lock-VWF (lower panels), indicating that the unfolded A2 domain may bind other VWF regions also, in addition to homotypic association. *P < .05 with respect to all other treatments at indicated times. Data are from 3 repeats.