Role of VWF-A2 in self-association. (A) Schematic of VWF proteins. A1 domain is deleted in ΔA1-VWF. WT protein (WT-VWF) has vicinal Cys in the A2 domain. The disulfide bond links the N and C terminus of VWF-A2 in Lock-VWF. (B) Protein multimer distribution of VWF variants expressed in HEK293T-furin cells. (C) Time course of VWF cleavage by 1 U/mL ADAMTS13 in the presence of 1.6 M urea at 37°C. Minimal cleavage of Lock-VWF is noted. (D-E) Five × 10⁷/mL washed platelets were shear mixed at 9600/s in a cone-plate viscometer along with 10 μg/mL ΔA1-488, either in the absence of VWF or upon addition of 10 μg/mL WT-VWF or Lock-VWF. Buffer calcium concentration was varied from 0 to 1.5M CaCl₂ (EDTA was not added). VWF self-association (D) and percentage platelet activation (E) were measured using flow cytometry for samples withdrawn at 5 minutes. VWF self-association quantifies the percentage of platelets binding more than basal (t = 0) levels of Alexa 488-conjugated ΔA1-VWF (ΔA1-488). Platelet activation measures the percentage of platelets binding PE-conjugated Annexin V. (F-G) VWF self-association (F) and platelet activation (G) triggered by WT-VWF was blocked by 20 µg/mL mAbs AK2 (anti-GpIbα) and AVW-3 (anti-VWF A1 domain). Data in panels D-G are from 3 to 4 independents runs, each containing 3 technical replicates. *P < .05 with respect to all other treatments at that calcium concentration. VWF self-association and platelet activation are higher for runs performed with WT-VWF compared with Lock-VWF.