Kinin-free High Molecular Weight Kininogen Research Articles

Tc-99m Glu-Cys-Gly-His-Gly-Lys (ECG-HGK), a novel Tc-99m labeled hexapeptide for molecular tumor imaging.

Domain 5 of kinin-free high molecular weight kininogen inhibits the adhesion of many tumor cell lines, and it has been reported that the histidine-glycine-lysine (HGK)-rich region might be responsible for inhibition of cell adhesion. The authors developed HGK-containing hexapeptide, glutamic acid-cysteine-glycine (ECG)-HGK, and evaluated the utility of Tc-99m ECG-HGK for tumor imaging. Hexapeptide, ECG-HGK was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling efficiency was evaluated. The uptake of Tc-99m ECG-HGK within HT-1080 cells was evaluated in vitro. In HT-1080 tumor-bearing mice, gamma imaging and biodistribution studies were performed. The complexes Tc-99m ECG-HGK was prepared in high yield. The uptake of Tc-99m ECG-HGK within the HT-1080 tumor cells had been demonstrated by in vitro studies. The gamma camera imaging in the murine model showed that Tc-99m ECG-HGK was accumulated substantially in the HT-1080 tumor (tumor-to-muscle ratio = 5.7 ± 1.4 at 4 h), and the tumoral uptake was blocked by the co-injection of excess HGK (tumor-to-muscle ratio = 2.8 ± 0.6 at 4 h). In the present study, Tc-99m ECG-HGK was developed as a new tumor imaging agents. Our in vitro and in vivo studies revealed specific function of Tc-99m ECG-HGK for tumor imaging.

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

We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y. H., Ogikubo, O., Otsuka, T., and Ohkubo, I. (2001) Biochem. Biophys. Res. Commun. 288, 975-980). In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro. P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg. Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.

Cardiac bypass haemostasis: putting blood through the mill.

Cardiac bypass haemostasis: putting blood through the mill.

Contact activation factors in plasma from women using oral contraceptives - increased levels of factor XII, kinin-free high molecular weight kininogen and acetone-activated kallikrein

The plasma levels of FXII, prekallikrein (PK) and high- and low molecular weight kininogens (HK and LK) were measured in women on low estrogen dose (30–40 μg ethinylestradiol) oral contraceptives (OC) and in controls. FXIIa was assayed in acetone-treated citrated plasma (CPL) with PK and the tetrapeptide S-2222 as substrates, and in acetone-treated citrated plasma with benzamidine (BPL) with PK as substrate. The level of FXII was found to be about 20% higher in OC-plasma than in control plasma. Kallikrein was assayed in CPL with S-2222 as substrate and in BPL with the tripeptide S-2302 as substrate. No difference in PK-level was observed in the CPL-based method, whereas an increase in kallikrein activity of about 30% was registered in BPL. The levels of HK and LK were estimated both in rocket immunoassay and in bioassay of released kinin. No difference in HK-level could be registered in immunoassay, whereas the bioassay revealed a HK-level in OC-plasma of 40% of the control level. Immunoblot studies showed that a substantial part of HK in OC-plasma was present as kinin-free protein (mol. wt. 103 KD), assumed to possess a higher cofactor potency than that of native HK. Both in bioassay and immunoassay the level of LK was found to be 60% higher in OC-plasma than in control plasma. Considered together the observations on contact factors made in this study provide support for the assumption of an increased readiness for contact activation in plasma from women using OC.

Characterization of guinea-pig high-molecular-weight kininogen as multi-functional molecule

High-molecular-weight (HMW) kininogen was purified from guinea-pig plasma by measuring its ability to correct the prolonged clotting time in human HMW kininogen deficient plasma (Fitzgerald trait). The purified HMW kininogen demonstrated a homogeneous band in disc gel electrophoresis in the presence of sodium dodecyl sulfate under reducing or non-reducing conditions with an apparent molecular weight of 100 000. Kinin released from HMW kininogen by treatment with guinea-pig plasma kallikrein was identified as bradykinin by reverse-phase HPLC and amino-acid analysis. The capacity of HMW kininogen as a thiol-proteinase inhibitor was realized by its dose-dependent inhibitory activity to papain. The K i value for papain was estimated to be 42 pM. The kinin-free HMW kininogen maintained the inhibitor and clotting-factor activities with similar capacities to those of the HMW kininogen molecule. Heavy chain (H-chain) and light chain (L-chain) of HMW kininogen were prepared from reduced and alkylated kinin-free HMW kininogen by HPLC. The S-alkylated H-chain, but not L-chain, demonstrated the inhibitor activity with the K i value 6.9 nM for papain, whereas the S-alkylated L-chain, but not H-chain, maintained the clotting activity one-third of the capacity of HMW kininogen. Specific antibodies recognized HMW kininogen, but also a probable low-molecular-weight kininogen(s) with an apparent molecular weight of 60 000 in the guinea-pig plasma. All of these properties are consistent with the reports on human, bovine and rat HMW kininogen.

Binding and dissociation of hageman factor, prekallikrein and high molecular weight kininogen in human plasma during contact activation

A kaolin pellet was incubated with human plasma at room temperature and immediately washed to remove all unbound proteins. Whereas in normal plasma, 154 mU(PPAN) of kallikrein was bound to the kaolin surface, in Hageman factor (HF)-deficient plasma or normal plasma preincubated with polybrene the surface-bound kallikrein was undetected, while a trace of prekallikrein was bound to the kaolin surface. Dissociation of kinin and kallikrein from the kaolin surface occurs during varying periods of incubation of the kaolin suspension alone. The dissociation of the surface-bound kallikrein revealed two phases in a 15 min incubation: the first phase of dissociation which rapidly progressed until the kinin liberation reached a plateau was followed by the second phase where the kallikrein was slowly dissociated and kinin was no longer liberated. Treatment of the kaolin suspension with trypsin, plasmin or plasma kallikrein enhanced the kinin liberation and dissociation of kallikrein from the kaolin surface. Kallikrein-kinin-free high molecular weight (HMW) kininogen-activated HF complex, kalli-krein-kinin-free HMW kininogen complex, kallikrein, kinin-free HMW kininogen and two activated HFs were found on Sephacryl S-300 and Sephadex G-100 gel filtrations of the supernatant obtained after a 60 min incubation of the kaolin suspension alone. The ternary complex of kallikrein, kinin-free HMW kininogen and activated HF suggests the presence of prekallikrein-HMW kininogen-HF complex on the kaolin surface.

Characterization of human high molecular weight kininogen. Procoagulant activity associated with the light chain of kinin-free high molecular weight kininogen.

Human high molecular weight (HMW) kininogen has been isolated and was found to be a single chain protein of approximately equal to 120,000 daltons. Upon digestion with plasma kallikrein bradykinin is generated, and SDS gel electrophoresis of the kinin-free protein reveals an apparent loss in size of 15,000 daltons. The kinin-free kininogen retains full activity as a coagulation factor and consists of two chains: a heavy chain of approximately equal to 66,000 daltons disulfide-linked to a light chain of 37,000 daltons. The heavy chain of HMW kininogen shares antigenic determinants with LMW kininogen and possesses no detectable coagulant activity. The isolated light chain is shown to be responsible for the coagulant activity of HMW kininogen and contains a unique antigenic determinant that distinguishes HMW kininogen from low molecular weight kininogen.

Effect of bovine high molecular weight kininogen and its fragments on fitzgerald trait plasma

Fitzgerald trait, a deficiency of high molecular weight (HMW) kininogen, is characterized by a prolongation of both the activated partial thromboplastin time (aPTT) and of the kaolin-activated euglobulin lysis time. To study the role of HMW kininogen in coagulation and fibrinolysis, highly purified bovine HMW kininogen, kinin-free HMW kininogen, a histidine-rich fragment of HMW kininogen, and low molecular weight (LMW) kininogen were obtained. The effects of these proteins on the aPTT and kaolin-activated euglobulin lysis time of Fitzgerald trait plasma were studied. It was found that whole bovine plasma and HMW kininogen had identical capacities to partially correct the prolonged aPTT. Kinin-free HMW kininogen had only 30% of the correcting capacity of intact HMW kininogen. LMW kininogen had no effect on the aPTT of Fitzgerald plasma. The histidine-rich fragment prolonged the aPTT of Fitzgerald plasma and of normal human plasma without affecting the one-stage prothrombin time. HMW kininogen also corrected the kaolin-activated euglobulin lysis time of Fitzgerald plasma. Kinin-free HMW kininogen and LMW kininogen had weak capacities to correct the Fitzgerald kaolin-activated euglobulin lysis time. The histidine-rich fragment significantly prolonged the kaolin-activated euglobulin lysis time of Fitzgerald and normal plasmas. The histidine-rich fragment appears to inhibit the activation of Hageman factor (Factor XII) by kaolin or ellagic acid. Our data suggest that intact bovine HMW kininogen corrects the deficiency of Fitzgerald trait plasma; but the hydrolysis of HMW kininogen by plasma kallikrein yields peptides with decreased correcting activity, and at least one with inhibitory activity in contact-mediated coagulation and fibrinolysis.