Rapid purification of human high molecular weight kininogen

Human high molecular weight kininogen was isolated by a rapid procedure, using anion exchange chromatography on QAE-Sephadex, ammonium sulfate precipitation and cation exchange chromatography on CM-Sephadex. The poor recovery and relatively low specific activity observed in earlier experiments was found to be due to a contaminant, presumably enzymatic, capable of releasing kinin from the kininogen. The spontaneous kinin release was blocked by soy bean trypsin inhibitor and by C1-inactivator. The isolated kininogen was stable at different temperatures, did not contain free kinin and was a good substrate for plasma kallikrein and plasmin.Supported by the Ontario Heart Foundation, the Medical Research Council of Canada and the Atkinson Charitable Foundation.Postdoctoral Fellow supported by the Ontario Heart Foundation.Research Associate of the Medical Research Council of Canada.     

Studies on the antigenicity and carbohydrates of human low molecular weight kininogen

Native low mol. wt (LM_λ) kininogen from human plasma and a kinin-free kinigogen from Cohn's plasma fraction IV (H_c-antigen) were isolated and studied with regards to their immunological reactivity and carbohydrate heterogeneity. Antisera were prepared against the conformational and sequence-dependent determinants of the heavy chain which is the common denominator of plasma kininogens. The molecules were characterized by single radial immunodiffusion and SDS gel electrophoresis. The effect of exoglycosidase treatment was investigated by a radioimmunoassay. It was found that the antigenic combining sites were not influenced by the partial removal of carbohydrates. These results suggest that the determinant structures of LM_λ kininogen reside in the peptide backbone of the protein. Carbohydrate heterogeneity was shown by different binding to concanavalin A and wheat germ lectin. With concanavalin A 52% of immunoreactive H_c-antigen was reactive compared to 76% of the native LM_λ kininogen. Th weak reactivity (11%) of the H_c-antigen towards the wheat germ lectin indicates that the N-acethyglucosamine residues are apparently blocked or missing. Immunoreactive kininogen was in all cases measured by single radial immunodiffusion or an RIA. The molecular nature of the heterogeneity is discussed in relation to the known isoelectric variations of kininogen.     

Conformation and sequence dependent antigenic determinants in human low molecular weight kininogen

Conformation and sequence-dependent antigenic determinants were investigated using a kinin-free low molecular weight kininogen isolated from Cohn's plasma fraction IV. This antigen contains the determinants of the apparently intact heavy chain common to the high molecular weight and low molecular weight kininogens. Straightforward reduction and carboxymethylation destroyed the immunoreactivity of this molecule. Antiserum prepared against the reduced protein recognized both reduced and unreduced antigen showing the presence of both types of antigenic determinant. The corresponding antibodies were separated using immunoadsorbent columns. As shown by the higher avidity of the antibodies, the conformation-dependent determinants dominate the antigenic structure.     

Bacterial adhesion to polyurethane surfaces in the presence of pre-adsorbed high molecular weight kininogen

The factors which affect the adherence of a bacteria cell to the surface of a biomaterial include the surface chemistry of the cell and material, as well as the composition of the adsorbed protein layer when the biomaterial is exposed to circulating blood. In an effort to better understand the mechanisms by which bacteria adhere to such surfaces, and specifically to determine the effects of high molecular weight kininogen on bacterial adhesion, experiments were performed in which the attachment of Staphylococcus aureus was directly observed on glass and on a series of functionalized polyurethanes. These surfaces had been pre-adsorbed with various concentrations of high molecular weight kininogen and fibrinogen. Attachment was observed using a radial flow chamber, in which shear stress varied inversely with radial distance. Protein adsorption studies were also performed using ¹²⁵I labeled fibrinogen to investigate the relationship between surface chemistry, protein adsorption, and bacterial attachment. Bacterial attachment was significantly decreased when the glass surface was pre-adsorbed with high molecular weight kininogen-either alone, or following adsorption of fibrinogen. High molecular weight kininogen thus exhibited anti-adhesive effects. On polyurethane surfaces pre-adsorbed with fibrinogen, kininogen, and albumin, the highest bacterial attachment was found on the base polyurethane, while significant decreases were seen on the hydrophilic polyurethanes. In addition, it was found that the surface with the least bacterial attachment and fibrinogen deposition was the polyurethane with pendant phosphonate groups.     

HKD5对人脐静脉内皮细胞游走及血管形成的抑制作用

目的:体外实验研究活化型高分子量激肽原（HKa）轻链富含组氨酸区域5 (HKD5)对人脐静脉内皮细胞（HUVECs）的粘附、游走及血管形成的影响。 方法:体外重组融合蛋白-活化型高分子量激肽原轻链富含组氨酸区域5 (GST-D5H)。WST-1法观察HUVECs细胞粘附能力；用改良Boyden Chamber膜侵袭系统观察HUVECs细胞游走(趋化)；用血管形成实验观察HUVECs细胞形成新生血管能力。 结果:GST-D5H作用后，HUVECs细胞粘贴率降低（P<0.05），游走穿膜细胞数明显低于对照组（P<0.01），诱导内皮细胞形成管腔数及长度均低于对照组（P<0.05）。 结论:GST-D5H能有效抑制HUVECs细胞粘附、游走，使该细胞粘附力降低，迁移性下降及血管形成数目减少或变细。     

Hageman factor, high molecular weight kininogen, and prekallikrein in chronic liver disease.

The activities of Hageman factor, high molecular weight kininogen (HMWK), and prekallikrein were studied in patients who had chronic active hepatitis and cirrhosis. Serum HMWK and prekallikrein activities were decreased in chronic active hepatitis and cirrhosis, but Hageman factor activity was low in cirrhosis only. The reduction of prekallikrein, HMWK, and Hageman factor was dependent on the degree of liver failure. Similar prekallikrein values were found in serum samples, activated or not, with an excess of Hageman factor and HMWK, which suggests that the decrease of prekallikrein in liver disease is not influenced by the simultaneous decrease of Hageman factor and HMWK.     

Interaction of high molecular weight kininogen with plasminogen inhibits binding of plasminogen to cell surfaces

Recent studies have shown that high molecular weight kininogen (HK) inhibits the binding of plasmin to platelets and thereby inhibits platelet activation by plasmin. We hypothesized that these results might be explained by HK binding to plasmin(ogen) in solution. Using ligand blotting, we showed that ¹²⁵I-plasminogen (Pg) binds to immobilized HK and that ¹²⁵I-HK binds to immobilized Pg. HK partially inhibited ¹²⁵I-Pg binding to lysine-Sepharose. Non-covalent binding of HK to Pg in solution was demonstrated using the cross-linking agent, bis(sulfosuccinimidyl)suberate (BS³) . The cross-linking agent stabilized a 180kDa complex as determined by SDS-PAGE. The 180kDa complex was not observed in the absence of BS³ or when ϵ-amino caproic acid was added. In addition, excess unlabelled Pg or HK inhibited the binding of each radiolabelled protein to the other. The equilibrium dissociation constant (K_D) for the binding of Pg to HK in solution was 0.8 μmol/L, as determined by BS³ cross-linking. Binding of Pg to C6 glioma cells in culture was inhibited by HK in a concentration-dependent manner with an IC₅₀ of approximately 0.4 μmol/L. Similar inhibition was observed using human umbilical vein endothelial cells (IC₅₀≈0.5 μmol/L). Binding of ¹²⁵I-HK to C6 glioma cells was apparently saturable, zinc-dependent and inhibited by Pg (IC₅₀≈0.9 μmol/L). The results presented here suggest that HK inhibits Pg binding to cell surfaces primarily by forming a complex with Pg in solution. Pg binding to HK may affect the localization of these proteins in the vascular compartment.     

Quantification of human high molecular weight kininogen by immunoblotting with a monoclonal anti-light chain antibody

Activation of the Hageman factor-dependent pathways in human plasma leads to the cleavage of high molecular weight kininogen (HMWK) into a disulfide-linked two-chain (heavy and light chain) molecule and release of bradykinin, a vasoactive peptide. We have utilized murine monoclonal antibodies to the light chain of HMWK (Blood (1988) 71, 1344) and developed a very sensitive immunoblotting assay to detect and quantify the amount of cleaved or uncleaved HMWK in whole plasma. The total HMWK content of plasma from apparently healthy donors was 55 micrograms/ml by this method. Cleaved HMWK was detected when only 2% of the plasma had been activated and the method was sensitive down to 2 ng of HMWK. Because of the extreme lability of bradykinin in body fluids, quantification of cleaved HMWK provides an important adjunct which reflects contact activation and permits calculation of a theoretical upper limit of the potential kinin formed.     

Adsorption from plasma and buffer of single- and two-chain high molecular weight kininogen to glass and sulfonated polyurethane surfaces

The adsorption of high molecular weight kininogen (HK) in its single-chain (SCHK) and two-chain (TCHK) forms from single protein solutions, plasma, and kininogen-deficient plasma, to glass and sulfonated polyurethane surfaces is reported. Using radiolabelling methods, it was found that in a single protein buffered system there was no difference in the adsorbed amounts of SCHK and TCHK over the concentration range 5-100 microg ml(-1) (similar to that in plasma). The adsorption of the two forms from normal plasma was also the same. However, immunoblots using an anti-HK antibody indicated that over the 2 h adsorption time, much of the SCHK present in the plasma was converted to TCHK: the band at 120 kD representative of intact SCHK disappeared, and bands at 56 and 46 kD representative of the heavy and light chains of TCHK were generated. To prevent conversion of SCHK to TCHK, the kallikrein inhibitor aprotinin (or in some cases a protease inhibitor cocktail), was added to the plasma in subsequent experiments. In addition, kininogen-deficient plasma was used (with either labelled SCHK or TCHK added) to avoid ambiguity in the tracer-population relationship. It was again found that there was no difference in the amounts of SCHK and TCHK adsorbed to glass and the sulfonated polyurethanes. The significance of these findings in relation to the reported anti-cell adhesion properties of adsorbed HK is discussed.     

High molecular weight kininogen inhibition of endothelial cell function on biomaterials

Synthetic vascular grafts implanted into humans fail to develop a complete endothelial lining. In previous studies, we have shown that high-molecular-weight kininogens (HMWK) adsorb to the surfaces of biomaterials. In addition, it has been demonstrated that these proteins modulate cellular function. In the present study, we report on the adhesion and proliferation of human umbilical-vein endothelial cells (HUVEC) on tissue culture polystyrene, glass, polyurethane, and Mylar(trade mark) surfaces coated with human HMWK, either single-chain HMWK (SC-HMWK) or double-chain HMWK (DC-HMWK). Surfaces coated with fibronectin served as a positive control for these experiments. Parallel experiments were performed in which HUVEC were allowed to migrate from crosslinked dextran microcarrier beads (Cytodex 2) onto HMWK-coated surfaces. Our results indicate that HMWK-coated surfaces inhibit endothelial cell adhesion, proliferation, and migration at 24 and 72 h, and this inhibition is concentration dependent. To determine a potential mechanism for this inhibitory phenomenon, cells were stained for cytoskeletal actin filaments using rhodamine-phalloidin. Endothelial cells on HMWK-coated surfaces displayed F-actin filament reorganization/disassembly, characterized by the absence of peripheral actin bands in focal adhesion contacts. We conclude that HMWK inhibit endothelial cell adhesion, proliferation, and migration on a variety of biomaterial surfaces. This inhibitory effect may play a role in promoting the lack of endothelialization in synthetic vascular grafts, which is thought to play a significant role in the failure of these devices.     

A low molecular weight heparin in hemodialysis

Summary The purpose of our study was to check whether the dosage recommended for the low molecular weight heparin tested here, i.e., 50% of the corresponding unfractionated heparin dose, is adequate to prevent clot formation in the extracorporeal system. Sixteen dialysis treatments of 4–5 h were given to each of six chronic dialysis patients. In dialyses 1, 2, 15 and 16 unfractionated heparin (initial dose 35 IU/kg, continuous dose 20 IU/kg/h) was given, and in dialyses 3–14 low molecular weight heparin (initial dose 17.5 anti-Xa U/kg, continuous dose 10 anti-X U/kg/h). At these dose levels of low molecular weight heparin, clot formation occurred in the extracorporeal system in five of the six patients, despite the fact that the plasma anti-Xa level of 0.5 U/ml recommended by the manufacturer had been attained. For this reason the continuous dose of low molecular weight heparin had to be raised to approx. 80% of the corresponding continuous dose of unfractionated heparin. A plasma anti-Xa level of 0.7 U/ml is necessary to prevent extracorporeal clot formation.Abbreviations anti-Xa U Anti-factor Xa unit - aPTT Activated partial thromboplastin time - AT III Antithrombin III - IU International unit - LMWH Low molecular weight heparin - UFH Unfractionated heparin     

Surface activation of factor XII (Hageman factor) — Critical role of high molecular weight kininogen and another potentiator

When factor XII was adsorbed to kaolin it slowly became activated and converted prekallikrein to kallikrein. In the presence of HMW-kininogen the rate of activation of factor XII and consequently that of prekallikrein was markedly enhanced. The enhancing effect of HMW-kininogen was a dose-dependent phenomenon. In order to enhance the activation of factor XII on a surface the HMW-kininogen molecule had to be intact. Cleavage of HMW-kininogen by kallikrein decreased the enhancing effect of HMW-kininogen, there being an inverse relation between the bradykinin-generated and the capacity to enhance factor XII activation. Another potentiator of factor XII activation was isolated from proteins adsorbed to aluminum hydroxide. This potentiator further increased the activation of factor XII, also in a dose-dependent fashion. It was postulated that factor XII is slowly converted into its active form by exposure to negatively charged surfaces; that this process is enhanced by kallikrein and further accelerated by HMW-kininogen and the potentiator; and that these enhancing substances probably act by opening active sites on the factor XII molecule.Supported by a grant from the Ontario Heart Foundation.Supported by the Medical Research Council of Canada.     

New congenital deficiency of high molecular weight kininogen and prekallikrein (Fitzgerald trait). Study of response to DDAVP and venous occlusion

The prolonged partial thromboplastin time observed in the plasma of a 36 year old asymptomatic man was related to the reduced prekallikrein activities (coagulant; antigenic; and amidolytic) and the absence of coagulant and immunologic activities of high molecular weight kininogen (HMWKg). The patient's plasma also exhibited impaired surface-mediated fibrinolysis and impaired generation of kallikrein. The coagulation defect was identified as the "Fitzgerald trait". The levels of CH50, C2, C4 and C-1 inactivator were normal. Venous occlusion in the patient gave rise to a normal release of extrinsic plasminogen activator from the vascular endothelium. The administration of DDAVP led to a FVIII/VWF response which was similar to that obtained in healthy subjects. No alteration could be observed in the contact phase proteins after DDAVP administration.     

Phylogeny of low molecular weight immunoglobulins

Because comprehensive studies of antigenic comparison of LMW Igs of non-mammalian vertebrates are lacking, such investigations have been done using double antibody RIA inhibition methods and antisera, produced in rabbits and carps. The dominant 7S Igs of anuran amphibians, reptiles, and birds are highly cross-reactive among one another and therefore belong to one class. They are different from mammalian IgG and IgD. In contrast to the so-called chicken IgA which shows no antigenic cross-reactivity with mammalian IgA and should be named IgB, the dominant 7S Igs of amphibians, reptiles, and birds have a well defined antigenic similarity with mammalian IgA. For that reason they do belong to the IgA class. A phylogenetic tree of Igs is discussed.     

Heat-mediated,ultra-rapid electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes

Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electro-transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins.