Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser 166 of glycoprotein Ib(beta).
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 GPIbbeta is phosphorylated at Ser(166) by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbbeta phosphorylation, a GPIb-IX mutant replacing Ser(166) of GPIbbeta with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbbeta (Delta165) 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(166) phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Delta165 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 GPIbbeta at Ser(166) negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D016466:CHO Cells; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D017353:Gene Deletion; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D014162:Transfection; D014841:von Willebrand Factor / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases