Platelet mechanotransduction by beads bearing immobilized VWF and anti-GpIbα mAb. A total of 107 platelets/mL were mixed with beads bearing either pVWF (A-B,D) or mAbs AP-1 against GpIbα (C,E) at various shear rates. Platelet:bead ratio was approximately 5:1. Anti-GpIbα blocking mAb AK2 was present in panel B, whereas there were no blocking reagents in panels A and C. Samples withdrawn at indicated times were incubated with CD31 PerCP-eFluor 710 and annexin-PE for 5 minutes and analyzed using flow cytometry. Cytometry forward versus side scatter was used to gate either singlet beads (Ai,Bi,Ci) or singlet platelets (Aii,Bii,Cii). (D-E) Distribution of active platelets that were either free (Aii,Bii,Cii top right quadrant) or bound (Ai,Bi,Ci top right quadrant) to beads was quantified. Percentage is based on total number of platelets in the sheared mixture read in cytometer. Beads without platelets appear in bottom left quadrant of Ai, Bi, Ci. *Platelet activation is higher at 3 minutes compared with 0 minutes at these shear rates (P < .05).
