The Study of GPIb-VWF Mediated Early-Stage Platelet Activation Triggering On a Single Cell

Abstract 1069

Binding of GPIbα to VWF tethers platelets to disrupted vascular surface during the haemostatic process. The GPIbα –VWF interaction can also trigger outside-in signaling cascade, resulting in platelet activation, characterized by morphological transformation from discoid to a more spiky shape as well as activation of integrin α _IIbβ₃. Using the adhesion frequency assay with a biomembrane force probe (BFP), we studied signal initiation by repeated brief contacts of a single platelet with a glass bead coated with VWF-A1 domain and/or fibronectin III 7–10 domain (FNIII_7–10) in a precisely controlled fashion (Fig. A). Contacting platelets with beads coated VWF-A1 only resulted in adhesion kinetics mediated by GPIbα –VWF interaction kinetics independent of the activation stage of the platelet. Contacting platelets with beads coated FNIII_7–10 only resulted in adhesion kinetics that correlated with the activation stage of the platelet. Discoid-shaped platelets yielded low level adhesions mediated by FN interaction with inactive α _IIbβ₃ (Fig. B, blue). By comparison, spiky-shaped platelets produced high level adhesions mediated by FN interaction with activated α _IIbβ₃ (Fig. B, red)that was four times stronger than the interaction with inactive α _IIbβ₃. Contacting platelets with beads coated both VWF-A1 and FNIII_7–10 resulted in two-stage adhesion kinetics. The first stage was mediated by GPIbα –VWF binding, which triggered a second stage consisting of an increase in adhesion after a sub-second delay. The second-stage binding coincided with morphological changes characteristic of platelet activation and matched that mediated by FN interaction with activated α _IIbβ₃. On the other hand, the concurrent calcium imaging showed as the platelet target was brought to the A1 bead in a repeating manner, the recorded calcium fluorescence intensity climbed up as the repeated touches continue (Fig. C). The peak temporally correlates with the morphological change. Our data indicates that binding of VWF-A1 to platelet GPIbα initiates outside-in signaling, leading to rapid irreversible platelet shape changes and calcium mobilization within a few seconds.

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