Inhibition of Platelet Adhesion and Aggregation by a Defined Region (Gly-486–Lys-502) of High Molecular Weight Kininogen

Abstract

Proteolytic cleavage of single chain high molecular weight kininogen (HK) by kallikrein releases the shortlived vasodilator bradykinin and leaves behind twochain high molecular weight kininogen (HKa). HKa and particularly its His-Gly-Lys-rich domain 5 have been previously reported to exert anti-adhesive properties by binding to the extracellular matrix protein vitronectin (VN). In this study the ability of HKa and domain 5 to interfere with platelet adhesion and aggregation was investigated. In a purified system HKa and particularly domain 5 but not HK inhibited the binding of VN to the ␣ IIb ␤ 3 integrin, whereas the binding of fibrinogen to this integrin was not affected. The region Gly-486 -Lys-502 from the carboxyl terminus of the domain 5 was identified as responsible for inhibition of the VN-␣ IIb ␤ 3 -integrin interaction, as this portion was also found to mediate kininogen binding to VN. Through these interactions, HKa, the isolated domain 5, and the peptide Gly-486 -Lys-502 abrogated the ␣ IIb ␤ 3 -integrin-dependent adhesion of human platelets to VN but not to fibrinogen. The codistribution of VN and HKa at sites of ex vivo platelet aggregation was demonstrated by transmission immune electron microscopy, indicating that the described interaction is likely to take place in vivo. Moreover, domain 5 and the peptide Gly-486 -Lys-502 dose-dependently blocked platelet aggregation, resembling the inhibitory effect of monoclonal antibody 13H1 against multimeric VN. Finally, treatment of mice with isolated domain 5 resulted in a significantly prolonged tail bleeding time. Taken together, our data emphasize the inhibitory role of HK domain 5 on platelet adhesion and aggregation; new antithrombotic compounds may become available on the basis of peptide Gly-486 -Lys-502 of HK domain 5.After vascular injury, the adhesion of platelets to the exposed subendothelial extracellular matrix together with platelet aggregation are essential steps in primary hemostasis. Subsequent fibrin formation not only stabilizes the temporary thrombus but also seals the platelet plug against diffusion of platelet secretory products, such as clotting factors, cell stimulatory agonists, or regulatory growth factors (1). At high shear rates platelets predominantly stick to von Willebrand factor and collagen, whereas under low shear rate or static conditions several other extracellular matrix proteins, such as fibrin and vitronectin (VN) 1 mediate ␣ IIb ␤ 3 integrin-dependent platelet adhesion via their Arg-Gly-Asp (RGD) sequence. Furthermore, binding of different agonists, such as ADP, thrombin, or collagen induces signaling events ultimately activating the receptor function of ␣ IIb ␤ 3 integrin for soluble fibrinogen leading to platelet aggregation (2-6).Because of its distribution between the flowing blood and the underlying tissues, VN is believed to exhibit several regulatory functions, particularly during vascular remodeling (7,8). The multimeric form of VN becomes incorporated into the vascular extracellular matrix (9, 10), accumula...

Effect of His-Gly-Lys Motif Derived from Domain 5 of High Molecular Weight Kininogen on Suppression of Cancer Metastasis Both in Vitro and in Vivo

“…It has been reported that the His-Gly-Lys-rich region of D5 H is able to bind to negatively charged surfaces or heparin as well as to cell surface proteoglycan (11,20,21). HKH20 (His 479 -His 498 ) peptide or HK486 (His 486 -Lys 502 ) peptide derived from D5 H , both include a His-Gly-Lys-rich region, has also been reported to inhibit the enzyme activity of plasminogen activator inhibitor-1 (23), adhesion and aggregation of platelets (24), and assembly of factor XII, prekallikrein, and HK (25).…”

“…The amounts of peptides per mouse were 500 g. On day 14, mice were sacrificed under anesthesia, and the metastatic colonies in lungs were counted. (24,26,27,45). The His-Gly-Lys-rich region (His 475 -Lys 502 ) of D5 H is thought to bind to a negatively charged region of the cell surface, and this region might be responsible for inhibition of cell adhesion.…”

Dietary oil modifies the plasma proteome during aging in the rat

“…Some studies suggest that an age-related increase in serum kininogens might play a role in modulating the changes in proliferative capacity of blood-exposed cells, including endothelial cells (Torres et al 2001). The proteolytic processing of kininogen involves the release of the nonapeptide bradykinin, a vasoactive and pro-inflammatory mediator, while the rest of the molecule has anti-adhesive properties (Asakura et al 1992) inhibiting platelet aggregation (Chavakis et al 2002). Therefore, the decrease of T-kininogen 1 after intake of a diet rich in VOO in both age groups could contribute to the decline of an inflammatory state.…”

A Novel Antithrombotic Role for High Molecular Weight Kininogen as Inhibitor of Plasminogen Activator Inhibitor-1 Function

“…clot retraction, an effect mainly mediated by domain 2 of HK (64). (iii) HKa interferes with both ligand binding to ␣ IIb ␤ 3 -integrin as well as ␣ IIb ␤ 3 -integrin-induced signaling (65). (iv) The binding of HKa to the urokinase receptor (66) on vascular cell surfaces may approximate kallikrein and prourokinase and thereby potentiate plasmin formation.…”