Identification and Characterization of Integrin alphaIIbbeta3 Intermediate Affinity State Induced By Gpibalpha Mechanotransduction

During arterial haemostasis, platelets tether to and translocate on disrupted vascular surface via binding of GPIbα to the VWF A1 domain, which triggers activation signals that induce intracellular Ca²⁺ release and up-regulate integrin α_IIbβ₃ binding capacity^1,2. Inhibition of this signaling pathway or blockade of α_IIbβ₃ binding has a devastating impact on platelet firm adhesion and aggregate formation^1,3,4.

We used a newly developed switch biomembrane force probe (BFP) assay to characterize the cooperativity between GPIbα and α_IIbβ₃ on single-molecular level. VWF-A1 and fibronectin fragment (FN_III7-10) were separately coated on two different probes, enabling controlled presentation of two ligands in separate space and time (Fig. A). Pulling GPIbα by A1 on the first probe with a durable force activated the discoid platelet (Fig. A, upper panel), as revealed by an intracellular calcium flux. Upon switching to the FN_III7-10-bearing bead, the A1-triggered platelet was interrogated for the activity of its surface α_IIbβ₃ (Fig. A, lower panel).

A 25-pN force of >2s duration on a single GPIbα-A1 bond was found to activate α_IIbβ₃ into an intermediate affinity state, as reflected by a ~30% adhesion frequency with a FN_III7-10 bead, significantly higher than binding to low affinity α_IIbβ₃ on platelets without A1-triggering but much lower than binding to high affinity α_IIbβ₃ on platelets pre-incubated with ADP or thrombin (Fig. B), two strong platelet-activating agonists. This submaximal activation agreed with the previous immuno-staining findings².

α_IIbβ₃ activation and outside-in signaling require the sequential engagement of talin and Gα₁₃ to β₃ cytoplasmic tail, respectively^5,6. Blocking Gα₁₃-β₃ engagement by a synthetic peptide mP₆ (gift from Xiaoping Du, UIC)⁶ had no effect on FN_III7-10 binding of resting and A1-triggered platelets, but significantly suppressed the FN_III7-10 binding of platelets pre-incubated with ADP or thrombin by lowering their adhesion frequencies to a level comparable with A1-triggered platelets (Fig. B). The control peptide mP₆Scr showed no effect on the ADP or thrombin stimulated platelets (Fig. B).

In addition to binding affinity, bond lifetimes were measured by force-clamp experiment. Platelets with no treatment, A1 triggering and ADP stimulation formed α_IIbβ₃-FN_III7-10 (Fig. C) and α_IIbβ₃-fibrinogen (Fig. D) bonds with short, intermediate and long lifetimes, respectively. Interactions with fibrinogen exhibited catch-slip bonds regardless of the α_IIbβ₃ affinity state. By comparison, only high affinity α_IIbβ₃ exhibited a catch-slip bond with FN_III7-10 as slip-only bonds were observed for FN_III7-10 interactions with intermediate and low affinity α_IIbβ₃. These data suggest the existence of three distinct integrin states that correspond to the three affinities and force-dependent bond lifetime patterns, in which the intermediate state was induced by the GPIbα mechanotransduction (Fig. E). Gα₁₃ engagement was not required for the intermediate state, but necessary for the high affinity state (Fig. E).

 

References:

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2. Kasirer-Friede, A. et al. Blood 103, 3403-3411, doi:10.1182/blood-2003-10-3664 (2004).

3. Savage, B., Saldivar, E. & Ruggeri, Z. M. Cell 84, 289-297 (1996).

4. Warwick, S. N. et al. Nature Medicine 15, doi:10.1038/nm.1955 (2009).

5. Gong, H. et al. Science 327, 340-343, doi:10.1126/science.1174779 (2010).

6. Shen, B. et al. Nature 503, 131-135, doi:10.1038/nature12613 (2013).