Regulation of Glycoprotein Ib-IX-von Willebrand Factor Interaction by cAMP-dependent Protein Kinase-mediated Phosphorylation at Ser of Glycoprotein Ib

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 GPIb is phosphorylated at Ser by cAMPdependent protein kinase (PKA). To understand the physiological role of GPIb phosphorylation, a GPIb-IX mutant replacing Ser of GPIb with alanine (S166A) and a deletion mutant lacking residues 166–181 of GPIb ( 165) 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 Ser phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or 165 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 GPIb at Ser negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.