Application of ΔA1-488 to monitor VWF self-association. (A) CHO-furin cells were generated by stably transfecting furin-internal ribosome entry site-hrGFP into CHO cells. Both recombinant full-length human VWF (rVWF) and VWF lacking the A1-domain (ΔA1-VWF; ie, amino acids 1242-1479) were expressed in these cells. (B) Western blot shows the multimer distribution of recombinant proteins. (C-D) ΔA1-VWF was labeled with Alexa-488 to produce ΔA1-488. ΔA1-488 bound platelets in the presence of both 5 μg/mL plasma (C, pVWF) and recombinant full-length VWF (D, rVWF). Shear rate is 9600/s. ΔA1-488 binding to platelets is the result of VWF self-association. Binding was blocked by mAbs against GpIbα but not GpIIb-IIIa. (E-F) A total of 5 μg/mL pVWF was shear mixed with 5 μg/mL ΔA1-VWF at 9600/s in the presence or absence of anti-GpIbα/GpIIb-IIIa mAbs for the indicated time. Two samples were withdrawn. The total VWF bound to platelets was measured in one sample using FITC-conjugated rabbit anti–human VWF polyclonal Ab (E). Platelet activation was quantified in the other using annexin-FITC. Both pVWF binding and cell activation were partially reduced by ΔA1-VWF in a GpIbα-dependent manner. Shear protocol in all panels is identical to Figure 1. *P < .05 with respect to all other treatments except blocking with GpIIb-IIIa mAbs. †P < .05 with respect to all other treatments.