The GPIb alpha intracellular tail - role in transducing VWF- and collagen/GPVI-mediated signaling

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