Cytometry detection of VWF binding and platelet activation. A total of 7.5 × 106 human platelets/mL was sheared with Alexa-488–conjugated plasma VWF (pVWF-488) in the absence or presence of unlabeled pVWF in a cone-plate viscometer at 9600/s. While pVWF-488 and pVWF were fixed at 5 μg/mL and 15 μg/mL, respectively, in panels A to C, this was varied over a wider range in panels D and E. In all cases, samples withdrawn at indicated times were incubated with CD31 PerCP-eFluor 710 and annexin-PE for an additional 5 minutes before cytometry analysis. (A) VWF (pVWF-488 or pVWF) presence and time of shear application are indicated in individual panels. All panels have 5000 events/dots. pVWF-488 and annexin V binding increases the number of events in top quadrants (top left plus top right) and right quadrants (top right and bottom right), respectively. (B) Platelets with pVWF-488 quantifies percentage of platelets having more than basal (time = 0) green fluorescence in top quadrants. (C) Platelet activation is a measure of percentage of platelets binding annexin-PE in right quadrants. (D-E) Identical to panels B and C, respectively, except for the use of a wider concentration range of pVWF-488/pVWF. As seen, pVWF-488 binding and platelet activation are augmented on addition of unlabeled pVWF. This is blocked by 25 μg/mL anti-GpIbα but not anti–GpIIb-IIIa mAb. *P < .05 with respect to all other treatments except blocking with GpIIb-IIIa mAb alone.
