The GPIbα intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling.

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The GPIbα-VWF A1 domain interaction is essential for platelet tethering under high shear. Synergy between GPIbα and GPVI signaling machineries has been suggested previously, however its molecular mechanism remains unclear. We generated a novel GPIbα transgenic mouse (GpIbaΔsig/Δsig) by CRISPR-Cas9 technology to delete the last 24 residues of the GPIbα intracellular tail that harbors the 14-3-3 and phosphoinositide-3 kinase binding sites. GPIbαΔsig/Δsig platelets bound von Willebrand factor (VWF) normally under flow. However, they formed fewer filopodia on VWF/botrocetin in the presence of a aIIbb3 blocker, demonstrating that despite normal ligand binding, VWF-dependent signaling is diminished. Activation of GPIbαΔsig/Δsig platelets with ADP and thrombin was normal, but GPIbαΔsig/Δsig platelets stimulated with collagenrelated- peptide (CRP) exhibited markedly decreased P-selectin exposure and aIIbb3 activation, suggesting a role for the GpIba intracellular tail in GPVI-mediated signaling. Consistent with this, while hemostasis was normal in GPIbαΔsig/Δsig mice, diminished tyrosine-phosphorylation, (particularly pSYK) was detected in CRP-stimulated GPIbαΔsig/Δsig platelets as well as reduced platelet spreading on CRP. Platelet responses to rhodocytin were also affected in GPIbαΔsig/Δsig platelets but to a lesser extent than those with CRP. GPIbαΔsig/Δsig platelets formed smaller aggregates than wild-type platelets on collagen-coated microchannels at low, medium and high shear. In response to both VWF and collagen binding, flow assays performed with plasma-free blood or in the presence of aIIbb3- or GPVI-blockers suggested reduced aIIbb3 activation contributes to the phenotype of the GPIbαΔsig/Δsig platelets. Together, these results reveal a new role for the intracellular tail of GPIbα in transducing both VWF-GPIbα and collagen-GPVI signaling events in platelets.