Platelet glycoprotein 1b: A zinc-dependent binding protein for the heavy chain of high-molecular-weight kininogen

Domains 3 and 5 of high-molecular-weight kininogen (HK) have been shown to bind to platelets in a zinc-dependent reaction. However, the platelet-bindi ng proteins responsible for this interaction have not been identified. We h ave focused on the platelet-binding site for the heavy chain (domain 3), wh ich we approached using a domain 3-derived peptide ligand and isolated bind ing proteins by affinity chromatography. The domain 3-derived peptide, thro mbin, HK, factor XII, as well as anti body to glycocalicin (the N-terminal portion of the alpha chain of GPIb) recognized a protein at 74 kD. We also isolated the thrombin receptor (PAR 1) at 45 kD, however, none of the above -mentioned ligands bound to this protein. Isolation of platelet membrane pr oteins using a monoclonal anti-glycocalicin antibody column revealed the sa me HK binding protein at 74 kD, which was reactive with anti-GPIb and repre sents a GPIb fragment. By photoaffinity labeling, HK interacted with membra ne GPIb, which was then isolated in native form (135 kD) along with gC1qR, a ligand for the HK light chain. Finally, I-125-HK binding to platelets was significantly inhibited by the anti-GPIb antibody. These results suggest t hat the GPIb, alpha chain, a known thrombin binding protein, is also one of the zinc-dependent platelet membrane binding sites for HK domain 3.