Préparation et dosage immunonéphélémétrique du fibrinogène de lapin

Pure rabbit fibrinogen was prepared by a method involving two ammonium sulfate precipitations, one 2 M phosphate buffer precipitation, one DEAE cellulose chromatography and lastly one Sepharose 6 B chromatography. The aminoacid composition was determined and an immunonephelemetric assay was proposed. This assay allowed an accurate determination of fibrinogen concentration in a rabbit with inflammatory reaction.     

Isolation and characterization of a specific antibody population against human fibrinogen

A specific antibody population against human fibrinogen was isolated from a rabbit antiserum by affinity chromatography on fibrinogen-bound Sepharose gel. Using a sensitive competitive radioimmunoassay, the antibody population was found to recognize epitopes on native fibrinogen but crossreacted minimally with fibrinogen fragment D and an early plasmin-degraded fibrinogen A alpha-chain product, but not at all with fragment E or fibrinopeptides A and B. Fibrin monomers shared part of these epitopes. The antibody population crossreacted to a small extent with bovine, horse and baboon fibrinogens and not at all with fibrinogens from sheep, rat, pig, goat, guinea pig, dog and rabbit.     

Plasminogen activator-anti-human fibrinogen conjugate

A covalent conjugate between the plasminogen activator urokinase and polyclonal rabbit anti-human fibrinogen has been formed using the heterobifunctional coupling reagent N-succinimidyl 3-(2-pyridyldithio) propionate. The resultant urokinase-anti-human fibrinogen conjugate was separated from unreacted material by gel filtration. The conjugate exhibited amidase activity against the small chromogenic substrate pyroglutamyl-glycyl-arginine-p-nitroanilide as well as plasminogen activator activity in an assay employing plasminogen and the plasmin substrate D-valyl-leucyl-lysine-p-nitroanilide. Retention of antibody specificity for fibrinogen was demonstrated using an enzyme linked immunoassay procedure. The conjugate was found to have greater stability in human plasma than unconjugated urokinase.     

Enzyme-linked coagulation assay. III. Sensitive immunoassays for clotting factors II, VII, and X

The solid-phase clotting assay utilizing fibrinogen coated on the wells of a microtiter plate and peroxidase-fibrinogen in solution as a substrate for thrombin (enzyme-linked coagulation assay, ELCA) has been modified for use as an immunoassay. Direct inhibition of factors II, VII, and X by polyclonal (rabbit) antibodies and of factor X by monoclonal antibodies has been demonstrated at high dilution of these antibodies and detection of the specific factors using ELCA. Using plates coated with a second antibody (goat anti-mouse IgG) as well as fibrinogen, monoclonal antibodies to factors X and VII were measured by binding the active factor to the plate and detection of the bound factor using ELCA. The assay was very sensitive, permitting the detection of as little as 0.2 ng/ml (30 pg/assay) of monoclonal antibody, or less than 0.4 ng/ml (60 pg/assay) of factor Xa. When plates were coated with monoclonal antibody to factor X and fibrinogen, the assay permitted the identification of distinct epitope specificities for two monoclonal antibodies to factor X by distinct competition of the monoclonal antibodies added in the solution phase for binding of factor Xa to the plate. This assay could be applied generally for immunoassay of clotting factors, and could have application in general as an immunoassay amplification system.     

Binding of hyaluronic acid to mammalian fibrinogens

We have postulated that the interaction of hyaluronic acid (HA), an extracellular matrix glycosaminoglycan, with fibrin is important during the early stages of wound healing and inflammation (J. Theor. Biol. 119:219; 1986), and have demonstrated the specific binding of 125I-labeled HA to human fibrinogen (J. Biol. Chem. 261:12 586; 1986). To determine whether HA binding is limited to human fibrinogen, we tested the ability of fibrinogens from various mammalian species to bind 125I-HA using a dot-blot assay. Increasing amounts of fibrinogen were adsorbed to nitrocellulose, and incubated with 125I-HA in the presence or absence of a 100-fold excess of nonradiolabeled HA to assess specific binding. In three independent experiments, the amount of 125I-HA bound/mg fibrinogen was determined from the slope derived by linear regression analysis of specifically bound 125I-HA versus protein concentration. A Student's t-test was performed to determine whether the slopes were statistically greater than zero. HA binding was considered statistically significant when P less than 0.05 was obtained by this analysis. Rabbit and dog fibrinogens significantly bound HA in all three trials. Baboon fibrinogen demonstrated significant HA binding in two of three trials. Pig, sheep and goat fibrinogens bound HA significantly in only one of three trials, whereas horse, rat and cow fibrinogens did not bind HA significantly at all. We conclude that fibrinogen from mammalian species other than human can specifically bind HA. The ability of fibrinogen to bind HA appears to correlate with an evolutionary divergence that separated human, baboon, dog, rabbit and rat from cow, pig, horse, goat and sheep.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Continuous culture of normal adult mammalian hepatocytes exhibiting parenchymal functions.

Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [3H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tryosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics.     

Isolation of alpha-1-macroglobulin (alpha 1 M) and alpha-2-macroblogulin (alpha 2 M) from rabbit blood]

A purification process of rabbit alpha 1 M and alpha 2 M from plasma was described: first the platelets, the fibrinogen, the plasminogen and the low-density lipoproteins were eliminated; then alpha 1 M and alpha 2 M were purified by gel filtration on Sephadex G 200 and by chromatography on DEAE-cellulose. These purified materials were then isotopically labeled allowing the study of the proteins metabolism.     

The role of the carbohydrate moiety in the biologic properties of fibrinogen

The carbohydrate moiety of some glycoproteins influences their secretion and functional properties. We have examined the importance of the oligosaccharide chains of fibrinogen in this regard. Fibrinogen was labeled de novo by the addition to rabbit hepatocyte monolayer cultures of either 3H-amino-acids or [2-3H] mannose, in the presence or absence of tunicamycin, a potent inhibitor of glycosylation. Inhibition of glycosylation, which ranged from 75 to 80%, was determined by incorporation of [2-3H]mannose as quantitated by gel filtration. Synthesis and secretion of fibrinogen were quantitated by 3H-amino-acid incorporation, using anti-fibrinogen immunoaffinity column chromatography of medium and cell homogenates. Tunicamycin did not appreciably inhibit fibrinogen synthesis, as compared to a 30-40% inhibition of overall protein synthesis, determined by incorporation of 3H-amino-acids into trichloroacetic acid-precipitable material. There was no evidence that secretion of fibrinogen was impaired. Fibrinogen from medium was copurified by adding cold plasma fibrinogen as carrier. Nonglycosylated fibrinogen was found to be functional as demonstrated by incorporation of radioactivity into clots of the copurified material at a rate identical to that of glycosylated fibrinogen. When clotted in the presence of Ca2+ and Factor XIII, cross-linking of glycosylated and nonglycosylated fibrin was demonstrable on fluorography of sodium dodecyl sulfate-polyacrylamide gels, showing disappearance of gamma-chain and appearance of gamma-gamma-dimers.     

赤爱胜蚯蚓(Eisenia Foetida Sarigny)纤溶酶的研究Ⅱ药理学作用

对蚯蚓纤溶酶进行了哈白兔体内、体外溶栓、抗凝试验,分别以蛇毒抗栓酶和尿激酶不同剂量组作对照试验,均有显著的溶解血栓功能.另外,蚯蚓纤溶酶溶解天然血块,对纤维蛋白原含量及凝血时间影响的药理学试验,结果证明蚯蚓纤溶酶具有溶栓与抗凝作用.     

A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo

Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well‐characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA‐based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost‐effective method that can be used to prevent unnecessary animal experiments.     

Isolation and characterization of luffacylin,a ribosome inactivating peptide with anti-fungal activity from sponge gourd (Luffa cylindrica) seeds

A purification scheme involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, and ion exchange chromatography on CM-Sepharose and Mono S was employed to isolate a peptide with a molecular weight of 7.8kDa from sponge gourd seeds. The peptide, which was designated luffacylin, exhibited an N-terminal sequence with pronounced resemblance to that of the 6.5kDa arginine-glutamate rich polypeptide previously isolated from sponge gourd seeds. Luffacylin inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 140pM and reacted positively in the N-glycosidase assay for ribosome inactivating proteins. Luffacylin exerted anti-fungal activity against Mycosphaerella arachidicola and Fusarium oxysporum.     

Separation of the protective enzyme bleomycin hydrolase from rabbit pulmonary aminopeptidases

Bleomycin (BLM) hydrolase inactivates the BLM class of antitumor antibiotics and protects against BLM-induced pulmonary fibrosis. This enzyme is poorly characterized but believed to be an aminopeptidase B. In the present report, both BLM hydrolase and aminopeptidase B from rabbit pulmonary cytosol were retained by arginyl-Sepharose and BLM-Sepharose affinity columns, further suggesting that these two enzymes are similar. When, however, BLM hydrolase was purified over 1800-fold by using our newly developed high-speed liquid chromatography assay for BLM hydrolase coupled with fast protein liquid chromatography, we found that this partially purified BLM hydrolase preparation lacked aminopeptidase B activity. Furthermore, BLM hydrolase was completely separated, by using anion-exchange Mono Q chromatography, from all the aminopeptidases identified in rabbit pulmonary cytosol: one aminopeptidase B, two aminopeptidases N, and one aminopeptidase with both aminopeptidase B and aminopeptidase N activities. Pulmonary BLM hydrolase also had a higher molecular weight than pulmonary aminopeptidase B. In contrast to aminopeptidase B, BLM hydrolase was not activated by NaCl and was much less stable at 4 degrees C. In addition, bestatin was a potent inhibitor of aminopeptidase B but had little effect on BLM hydrolase, while leupeptin was a potent inhibitor of BLM hydrolase but was less effective against aminopeptidase B. Thus, pulmonary BLM hydrolase and aminopeptidase B have affinity for each other's substrate, but they are clearly distinct enzymes on the basis of charge characteristics, molecular weight, stability, and sensitivity to inhibitors and activators.     

Glycation of fibrinogen in diabetic patients: a practical colorimetric assay

We have developed a practical method for the measurement of glycated fibrinogen which combines purification by glycine precipitation with a nitroblue tetrazolium assay at 56°C in the presence of Zwittergent 3–14. This detergent, at a concentration of 10 g/l, was chosen because it combines solubilization of fibrinogen and low coloration of blanks while increasing the sensitivity of the assay. A positive correlation (r=0.85,P<0.02) was found between this procedure and a previously reported one based on the thiobarbituric acid assay. To validate the method we then measured fibrinogen, glycated fibrinogen, serum fructosamine and erythrocyte glycated haemoglobin in a population of healthy euglycaemic subjects (n=30) and a population of diabetic patients (n=40). Glycated fibrinogen was significantly higher in diabetic patients than in control subjects (15.42±0.70vs 11.52±0.27 nmol of deoxymorpholinofructose equiv. per mg,P<0.005). Given the short half-life of fibrinogen, assay of its glycation may be useful as a short-term marker of glycaemic control.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Abstract Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using ¹²⁵I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cell. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant ( K _d) of 5.2 × 10⁻⁸ M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one compotent of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Structures involved in the binding of human fibrinogen to Candida albicans germ tubes

Recent evidence for the interaction between human fibrinogen and Candida albicans germ tubes have led us to attempt to characterize the structures involved. Using 125I-radiolabeled proteins, fibrinogen purified by affinity chromatography and its plasmin degradation products, the binding sites on the fibrinogen molecule appeared to be located specifically in the D-domain. Conversely to the fibrinogen and the fragment D, radiolabeled fragment E, however, did not interact with cells. The binding was time-dependent, saturable and reversible. Scatchard analysis of the data obtained revealed an average of 6000 binding sites per germ tube with dissociation constant (Kd) of 5.2 X 10(-8) M. No potent competition was observed for a range of different proteins and carbohydrates. Fibrinogen fragment D binding proteins were identified using a dithiothreitol-iodoacetamide extract of the fungus. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, one component of 68 kDa was detected. Thus, the presence of fibrinogen binding proteins specifically localized on the cell wall surface of C. albicans germ tubes may constitute one of the factors involved in the development of candidosis.     

Continuous culture of normal adult mammalian hepatocytes exhibiting parenchymal functions

Summary Past in vitro studies of liver-cell functions have been performed on nonproliferating primary cells or serially propagated hepatic monolayers of neoplastic or fetal origin. We optimized conditions for the selective culture of adult rabbit and canine liver parenchymal cells and presently have four differentiated proliferating monolayer strains. At the 30th passage level these hepatic cultures still display the specific liver parenchymal functions of albumin and fibrinogen synthesis as well as tyrosine aminotransferase activity. Optimization of the conditions for hepatocyte culture was monitored by [³H]thymidine incorporation. Albumin and fibrinogen synthesis were measured by bioradioimmunoassay and tyrosine transaminase activity by a modification of Diamondstone's assay. Albumin and fibrinogen synthesis were correlated with hepatocyte growth kinetics. Supported by the Medical Staff Research and Education Fund, Wayne County General Hospital, Eloise, Michigan 48132, and American Cancer Society Institutional Grant No. 40M, The University of Michigan, Ann Arbor, Michigan 48109.     

The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays

An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.     

Fibrinogen has chaperone-like activity

Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.