VWF self-association in the presence of plasma proteins and in whole blood. (A) A total of 30 × 106 platelets/mL was diluted in 100% VWF-deficient plasma, instead of HEPES buffer, along with 10 μg/mL FITC-conjugated pVWF (pVWF-FITC). The sample was sheared at 9600/s for 4 minutes, either in the presence or absence of 10 μg/mL unconjugated pVWF and anti-GpIbα blocking mAb HIP1. A total of 60 μg/mL 7E3 was present during all runs to prevent VWF interaction with GpIIb-IIIa. Samples withdrawn were incubated for 5 minutes with CD31-PE and annexin-PE/Cy5 before cytometry analysis. *P < .05 with respect to all other treatments. (B) A total of 5 μg/mL ΔA1-488 (green) was added to PRP and sheared at 9600/s for 2 minutes. Samples fixed in 0.5% paraformaldehyde were labeled with VM16d (anti-GpIbα mAb, red) and examined using confocal microscopy. (C) A total of 5 μg/mL ΔA1-488 was added to whole blood and subjected to shear at 4700/s. Samples withdrawn at 4 minutes was incubated with CD31 PerCP-eFluor 710 to identify platelets and annexin-PE to monitor cell activation. Cytometry dot plot shows ΔA1-488 bound to inactive (top left quadrant) and activated platelets (top right). (D) Distribution of active platelets either with or without ΔA1-488 bound. Percentage is based on total number of platelets read in the cytometer. *Platelet activation (top right plus bottom right) is higher at 4 minutes compared with 0 minutes at this shear rate (P < .05). †Platelet activation at 4 minutes at this shear rate is higher compared with 300/s (P < .05).
