Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser¹⁶⁶ of glycoprotein Ibβ

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates initial platelet adhesion and activation. It is known that the cytoplasmic domain of GPIbβ is phosphorylated at Ser¹⁶⁶ by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbβ phosphorylation, a GPIb-IX mutant replacing Ser¹⁶⁶ of GPIbβ with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbβ (Δ165) were constructed. These mutants, expressed in Chinese hamster ovary (CHO) cells, showed an enhanced VWF-binding function compared with wild type GPIb-IX. Treatment of CHO cells expressing wild type GPIb-IX with a PKA inhibitor, PKI, reduced Ser¹⁶⁶ phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Δ165 mutants showed a significantly enhanced adhesion to immobilized VWF under flow conditions. Consistent with the studies in CHO cells, treatment of platelets with PKI enhanced VWF binding to platelets. In contrast, a PKA stimulator, forskolin, reduced VWF binding and VWF-induced platelet agglutination, which was reversed by PKI. Thus, PKA-mediated phosphorylation of GPIbβ at Ser¹⁶⁶ negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.