Affinity For Fibrinogen Research Articles

Application of immunoperoxidase method to electron microscopic observation of plasma protein on polymer surface.

The immunoperoxidase technique was applied to observe the electron microscopic presence of human plasma protein on a polymer surface. Two types of polymer material, i.e., Dacron and a newly synthesized heparinized hydrophilic polymer (H-RSD, Toray Industries, Inc.), showed different reactions to human plasma proteins. The hydrophobic Dacron showed a decreased affinity for plasma albumin, but a greater affinity for fibrinogen, alpha 1-lipoprotein, and beta-lipoprotein when compared to H-RSD. The thrombo-resistant H-RSD showed a relatively strong affinity for albumin, but in a spotty or focal manner. Most of the H-RSD surface did not adsorb albumin. Fibrinogen adsorption was observed in areas smaller than albumin adsorption. The adsorption of fibrinogen appeared to occur through certain material. The alpha 1-lipoprotein and beta-lipoprotein were not adsorbed on the H-RSD. The immunoperoxidase method is a sensitive and useful technique in studying the interactions between plasma proteins and the surface of polymer materials.

Distribution of Plasma Fibronectin (Cold-Insoluble Globulin) and Components of the Factor VIII Complex After Heparin-Induced Precipitation of Plasma

AbstractFactor VIII procoagulant (VIII:C) activity, factor VIII coagulant antigen (VIII:CAg), von Willebrand ristocetin cofactor (VIIR:RC) activity, factor VIII-related antigen (VIIIR:Ag), and plasma fibronectin (CIg; cold-insoluble globulin) were measured in the heparin precipitable fraction (HPF) and heparin supernatant fraction (HS) of normal human plasma. Following heparin induced precipitation, most measurable VIII:C activity (77% ± 24%) was recovered in the HS. Although there was little VIII:C activity (<1%) in the HPF, 20% ± 6.5% VIII:CAg was present as well as CIg (81 % ± 5.6%), VIIIR:RC activity (72% ± 12%), and VIIIR:Ag (34 ± 5.2%). As assessed by Na dodecyl SO4 glyoxyl agarose electrophoresis, the multimeric forms of plasma VIIIR:Ag could be resolved into a series of bands. Larger multimers tended to precipitate with the HPF whereas the smaller multimers tended to remain supernatant. Plasma from a subject with congenital afibrinogenemia was also studied. Although the afibrinogenemic HPF contained CIg, neither VIIIR:RC activity nor VIIIR:Ag was precipitated. However, both were present in the HPF from afibrinogenemic plasma to which fibrinogen had been added, suggesting that they are incorporated in this precipitate because of an affinity for fibrinogen. The ability of heparin to induce precipitation of CIg while leaving most VIII:C activity in the supernatant plasma may be useful in the preparation of procoagulantrich plasma subfractions, since VIII:C can subsequently be recovered in good yield by cryoprecipitation.

Distribution of plasma fibronectin (cold-insoluble globulin) and components of the factor VIII complex after heparin-induced precipitation of plasma

Factor VIII procoagulant (VIII:C) activity, factor VIII coagulant antigen (VIII:CAg), von Willebrand ristocetin cofactor (VIIIR:RC) activity, factor VIII-related antigen (VIIIR:Ag), and plasma fibronectin (CIg; cold-insoluble globulin) were measured in the heparin precipitable fraction (HPF) and heparin supernatant fraction (HS) of normal human plasma. Following heparin induced precipitation, most measurable VIII:C activity (77% +/- 24%) was recovered in the HS. Although there was little VIII:C activity (less than 1%) in the HPF, 20% +/- 6.5% VIII:CAg was present as well as CIg (81% +/- 5.6%). VIIIR:RC activity (72% +/- 12%), and VIIIR:Ag (34 +/- 5.2%). As assessed by Na dodecyl SO4 glyoxyl agarose electrophoresis, the multimeric forms of plasma VIIIR:Ag could be resolved into a series of bands. Larger multimers tended to precipitate with the HPF whereas the smaller multimers tended to remain supernatant. Plasma from a subject with congenital afibrinogenemia was also studied. Although the afibrinogenemic HPF contained CIg, neither VIIIR:RC activity nor VIIIR:Ag was precipitated. However, both were present in the HPF from afibrinogenemic plasma to which fibrinogen had been added, suggesting that they are incorporated in this precipitate because of an affinity for fibrinogen. The ability of heparin to induce precipitation of CIg while leaving most VIII:C activity in the supernatant plasma may be useful in the preparation of procoagulant-rich plasma subfractions, since VIII:C can subsequently be recovered in good yield by cryoprecipitation.

Anticoagulant and fibrinolytic properties of salivary proteins from the leech Haementeria ghilianii.

AbstractThe salivary glands of the blood-sucking leech Haementeria ghilianii contain an anticoagulant that not only inhibits the clotting of human and bovine plasma, but also dissolves previously formed fibrin clots. This anticoagulant activity is attributable to an enzyme, for which the name hementin is proposed. Hementin catalyzes the proteolytic degradation of fibrinogen and fibrin, even in the presence of the inhibitors of proteases occurring in human plasma. The enzyme has the same affinity for human fibrinogen and fibrin. In human fibrinogen cleaves the Act chain initially and then the γ chain to yield characteristic fragments of high molecular weight that are different from the fragments resulting from the digestion of fibrinogen by plasmin. The salivary extracts do not contain any appreciable amounts of an activator of human plasminogen or an inhibitor of human or bovine thrombin. Thus, H. ghilianii prevents coagulation of its host blood through a fibrinogenolytic mechanism that is entirely differ...

Adsorption of proteins from plasma to a series of hydrophilic-hydrophobic copolymers. I. Analysis with the in situ radioiodination technique.

The adsorption of proteins affects cellular interactions with foreign surfaces and thus plays an important role in determining the biocompatibility of implants. Previous studies have indicated differences in the affinity of various proteins for a given polymer, and differences in the affinity of fibrinogen for a series of polymers varying in hydrophilicity. These studies suggest that differences in the composition of the protein layer adsorbed to polymers from plasma might exist. To examine this hypothesis, the proteins adsorbed from plasma to a series of polymers varying in hydrophilicity were analyzed with the iodogram technique. Copolymers of hydroxyethyl methacrylate and ethyl methacrylate made by the radiation grafting technique were exposed to plasma for 0.5 or 150 min. The adsorbed proteins were iodinated, eluted with SDS, and separated with polyacrylamide gel electrophoresis. Fibrinogen, immunoglobulin G, hemoglobin, and a peak tentatively ascribed to prothrombin were the major proteins detected. Very little iodine was incorporated into adsorbed albumin, even though it was shown to be present by a separate experiment using dye binding. The fraction of total radioactivity associated with each of nine proteins was found to vary markedly and systematically among the surfaces. The distribution of radioactivity into the proteins was very different on 0.5 and 150-min plasma exposed polymers. The results reflect both compositional differences in the adsorbed protein layer on the polymers and differences in the accessibility of proteins to the labeling reagent in the adsorbed state. Differences in the organization of the adsorbed protein layer may play a key role in determining whether cell surface receptors can come in contact with the specific plasma protein able to further stimulate the cell.

Affinity of fibrinogen and fibrin to collagen type III and denatured collagen type I demonstrated at low temperature

Human citrated plasma was passed at 4°C through columns of Sepharose 4B covalently coupled with native or denatured collagen, type I, or with native collagen, type III. Fibrinogen and cold-insoluble globulin were retained by immobilized denatured collagen, type I, and by native collagen, type III. At 20°C the two adsorbents only bound cold-insoluble globulin. Immobilized native collagen, type I, failed to retain fibrinogen even at low temperature but accumulated soluble fibrin created in plasma by limited thrombin incubation. There is evidence that the fibrinogen A α-chains are involved in the interaction with collagen, type III.

Relationship among cold isoluble globulin, fibrinogen, fibrin monomer and heparin in forming heparin precipitable fraction

The formation of heparin precipitable fraction (HPF) is often observed in the chilled plasma of patients with certain infections and inflammations that heparin had been treated. HPF is believed to consist of mainly fibrinogen and cold insoluble globulin (CIG). In this paper, to study the mechanism of formation of HPF in plasma we examined the relationship among CIG, fibrinogen, fibrin monomer and heparin in the formation of HPF.Heparin formed the precipitate from normal plasma in the cold at the optimal heparin concentration from 10 to 50Uperml of plasma. The further raising of the heparin level reduced the precipitate formation. The washed HPF contained CIG and fibrinogen by the analysis from the SDS-gel pattern of electrophoresis and the immunological test. Furthermore, the NH2-terminal analysis of HPF by direct Edman's method indicated the presence of alanine and tyrosine. These amino acids were corresponded to NH2-terminal residues of αand γ-chain of human fibrinogen but not to fibrin monomer, since NH2-terminus of either CIG and β-chain of fibrinogen were blocked with pyroglutamyl residue. From the experiments by using purified CIG and fibrinogen in the cold, heparin formed HPF with CIG alone or with mixture of CIG and fibrinogen but not with fibrinogen alone. From these results CIG is essential to form HPF. In the mixture composed of purified CIG, fibrinogen and heparin, Ca++ increased the formation of HPF at the optimal concentration from 10 to 50mM. Fibrinogen increased the amount of HPF and seemed to reduce the threshold level of CIG that need to form the precipitate.To clarify the interaction between CIG and fibrinogen or fibrin monomer, CIG was submitted to the chromatographic experiments with fibrinogen-agarose or fibrin monomer-agarose column. CIG had extremely higher affinity to bind the fibrin monomer than fibrinogen, and its affinity of CIG for fibrin monomer was completely disappeared by the addition of heparin (10U/ml) in the eluant. Furthermore, on the affinity chromatography of heparin-agarose column, CIG had a considerably higher affinity for heparin than fibrinogen did.From these results, it was indicated that CIG is essential factor in the formation of HPF in plasma, although fibrinogen participates mainly in HPF. And the participation of fibrinogen in HPF is seemed to occur by reason of its affinity for CIG through the small amount of fibrin monomer, because fibrin monomer has strong affinity for both fibrinogen and CIG.

Affinity chromatography of soluble fibrin in human plasma on insolubilized fibrinogen and on fragment D from fibrinogen, non cross-linked or cross-linked fibrin

Normal human plasma containing approximately 2.5 mg of fibrinogen per ml, 131l-labelled fibrinogen (less than 0.1 mg per ml) and 125l-labelled fibrin monomer (between 0.012 and 0.05 mg per ml) was subjected to affinity chromatography on insolubilized human fibrinogen (Fg-agarose), fragment D prepared from fibrinogen (FgD-agarose), fragment D from non cross-linked fibrin (FbD-agarose) or fragment D from cross-linked fibrin (D-D-agarose). The fibrin monomer was obtained by dissolving fibrin in 2 M NaBr. The fibrinogen and fragments D were highly purified materials which were coupled to CNBr-activated Sepharose in comparable molar amounts (8 mg per ml gel for the fragments D and 20 mg per ml gel for fibrinogen). The FgD- and D-D-columns had negligible affinity for fibrinogen in plasma, the Fg-column bound approximately 3 percent of the Fibrinogen and the FbD-column 1.5 percent. All columns showed very similar binding properties for soluble fibrin monomer both with respect to the amounts of fibrin adsorbed and the amounts eluted with 2 M NaBr. Thus the reported higher affinity of insolubilized fibrin monomer to fragment D prepared from fibrin compared to Fragment D from fibrinogen was not associated with a higher affinity of insolubilized fragments D from fibrin for soluble fibrin monomer.

Photooxidation of fibrinogen in the presence of methylene blue and its effect on polymerization

Human fibrinogen was illuminated in the presence of methylene blue. The resulting photooxidized fibrinogen was devoid of polymerization activity and thrombin-induced coagulability. The initial rate of the thrombin catalysed release of fibrinopeptides from photooxidized fibrinogen was normal. It was shown that illumination of photooxidized fibrinogen and photooxidized fragment N-DSK caused the modification of histidine residues. Tryptophan residues were also modified. When fibrinogen was photooxidized immediately after the addition of thrombin, the capacity to polymerize was lost. The inhibition of polymerization was less marked when oxidation was initiated at the time when polymerization began or thereafter. Photooxidized fibrinogen acts as an inhibitor of the polymerization of fibrin monomers. Photooxidized fibrinogen has affinity for thrombin-activated fibrinogen-Sepharose and thrombin-activated fragment N-DSK-Sepharose. When the former conjugate is illuminated in the presence of methylene blue its affinity for fibrinogen is decreased. It is concluded that the fragment N-DSK domain of fibrinogen is affected by photooxidation.

Isolation of fibrinogen from plasma by affinity chromatography on insolubilized fibrinogen (FG-ag) and insolubilized fibrin-monomer (FM-ag)

Insolubilized fibrinmonomer, which is converted from insolubilized fibrinogen by thrombin action, adsorbs soluble fibrinogen from solutions or plasma. After treatment with 6 molar urea without removal of the fibrinopeptides insolubilized fibrinogen gets a high affinity for soluble fibrinogen, too. Insolubilized fibrinmonomer and urea-treated insolubilized fibrinogen are suitable adsorbents for the purification of fibrinogen from plasma by affinity chromatography.