title: Human kininogens regulate thrombin binding to platelets through the glycoprotein Ib-IX-V complex.
abstract: We and others have shown that both high and low molecular mass kininogens are able to inhibit the thrombin-induced aggregation of gel-filtered platelets, indicating that the locus for inhibition resides in the heavy chain. The inhibitory site is present in domain 3, confined to the C-terminal portion of the region encoded by exon 7 (K270-G292), and the minimal effective sequence is a heptapeptide (L271-A277; Kunapuli et al, J Biol Chem 271:11228, 1996). Kininogens inhibit thrombin binding to platelets and thus inhibit thrombin-induced aggregation. The molecular mechanism by which kininogens inhibit thrombin-induced aggregation of platelets is unknown. Thrombin has previously been shown to bind to two receptors on the platelet surface, glycoprotein (GP) Ib-IX-V complex and the hepta-spanning transmembrane receptor coupled to G protein(s). We now show that, unlike its effect on normal platelets, kininogen (2 micromol/L) did not inhibit the thrombin-induced aggregation of Bernard-Soulier platelets, which lack the GP Ib-IX-V complex, suggesting that kininogen interacts either directly or indirectly with that complex and restricts access by thrombin to this receptor. We further show that both recombinant K270-G292 polypeptide and the synthetic peptide L271-A277 derived from high molecular mass kininogen lower thrombin binding to platelets in a manner similar to monoclonal antibodies to or ligands (von Willebrand factor and echicetin) of GP Ib-IX. The anti-GP Ib-IX-V complex antibodies, TM-60 and SZ 2, can inhibit 125I-high molecular mass kininogen binding to platelets. Conversely, kininogen could block the binding of biotinylated TM-60 or of 125I-SZ 2. Kininogen inhibited the binding of biotinylated thrombin bound to a mouse fibroblast cell line transfected with the GP Ib-IX-V complex. These results indicated that kininogen binds to the GP Ib-IX-V complex modulating thrombin binding to platelets and the consequent platelet aggregation. Kininogen can thus serve as an important regulator of the early stages of platelet stimulation by thrombin.
authors.affiliation: The Sol Sherry Thrombosis Research Center and Department of Medicine, Temple University School of Medicine, Philadelphia, PA 19140, USA.
authors.forename: H N
authors.initials: HN
authors.lastname: Bradford
authors.forename: R A
authors.initials: RA
authors.lastname: Dela Cadena
authors.forename: S P
authors.initials: SP
authors.lastname: Kunapuli
authors.forename: J F
authors.initials: JF
authors.lastname: Dong
authors.forename: J A
authors.initials: JA
authors.lastname: Löpez
authors.forename: R W
authors.initials: RW
authors.lastname: Colman
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001606:Bernard-Soulier Syndrome / Q000097:blood; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007704:Kininogens / Q000494:pharmacology / Q000502:physiology*; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D007704:Kininogens; D010446:Peptide Fragments; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; C108022:leucyl-asparaginyl-alanyl-glutamyl-asparaginyl-asparaginyl-alanine; D013917:Thrombin