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.
Animals Antibodies, Monoclonal