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N Heimburger

Publications and source records attributed to N Heimburger.

At least 37 records · Page 2Linked to original sources
PubMed1988

Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants.

Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.

Animals↗
PubMed1988

A new and simple isolation procedure for human protein C inhibitor. Evidence for a second inhibitor for activated protein C present in human plasma.

Human plasma contains an inhibitor of activated protein C (APC) which is termed according to its function protein C inhibitor (PCI). High purification of functionally active PCI with a yield of 18% is achieved by an improved procedure consisting of 4 steps: precipitation by rivanol, fractionation with ammonium sulfate, ion-exchange chromatography on DEAE Sephacel and chromatography on dextran sulfate Sepharose. This purification results in the isolation of a homogeneous PCI which migrates in immunoelectrophoresis with the beta-globulins of human plasma and in SDS PAGE as one single band at Mr = 57,000 both under reducing and nonreducing conditions. The specific activity of the highly purified PCI was determined to be 226 units/mg, 1 unit being equivalent to the activity of 1 ml fresh human citrated plasma. PCI forms complexes with 1:1 stoichiometry (Ki: 1.4 x 10(-8) M) resulting in a loss of the amidolytic activity of APC as measured on Tos-Glu-Pro-Arg-pNA (S 2366). The inhibition rate of APC by PCI (k: 7.5 x 10(5) M-1 min-1) is significantly increased in the presence of 5 i.u./ml heparin (kH: 2.2 x 10(7) M-1 min-1). PCI also blocks the amidolytic activities of urokinase plasminogen activator (u-PA), thrombin and factor Xa on their chromogenic substrates in a heparin-dependent manner. According to the Ki-values measured for these reactions PCI is a noncompetitive inhibitor of these proteases. The Ki-values calculated do not differ significantly from those obtained for the inhibition of APC by PCI. Immunodepleted PCI-deficient plasma still contains an inhibitory activity against APC which, however, only slowly inactivates the amidolytic activity of APC and in a time and concentration-dependent manner. Addition of heparin has no influence on the inhibition rate. This finding suggests the existence of a second, heparin-independent PCI present in human plasma.

Blood Proteins↗
PubMed1988

Determination of human thrombin-antithrombin III complex by enzyme immunoassay.

We have developed a specific and sensitive ELISA for the measurement of the TAT in human plasma. The assay follows the sandwich principle and uses two different antibodies directed against human thrombin and human antithrombin III, respectively. The anti-thrombin antibody population used for coating was purified by immunoadsorption on immobilized prothrombin and thrombin, respectively. Antithrombin III antibodies were conjugated with peroxidase. Plasma samples containing TAT were incubated in polystyrene tubes coated with anti-thrombin antibodies; after washing, peroxidase-conjugated antithrombin III antibodies were added and bound enzyme activity was subsequently measured using o-phenylenediamine. The assay was calibrated with definite concentrations (2.0 to 60 micrograms/l) of preformed purified TAT added to TAT-poor plasma. Plots of absorbance at 492 nm against TAT concentrations revealed a linear correlation (r = 0.98). A reference range from 0.85 to 3.0 micrograms/l was calculated from TAT concentration in plasma samples from 88 healthy donors (mean value +/- SD: 1.45 +/- 0.4 micrograms/l). In patients with deep vein thrombosis confirmed by phlebography (n = 15), TAT was found up to 7-13 micrograms/l. Patients with septicemia associated with a consumption coagulopathy (n = 10) showed markedly increased TAT values (greater than or equal to 10 micrograms/l). From these data it can be concluded that measurement of TAT might be a parameter for detection of a latent clotting pathway activation.

Antithrombin III↗
PubMed1987

Inhibition of urokinase by protein C-inhibitor (PCI). Evidence for identity of PCI and plasminogen activator inhibitor 3.

Human protein C-inhibitor (PCI) was isolated from human citrated plasma by combining rivanol precipitation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on dextran sulfate Sepharose. The purified PCI migrated with the beta-globulins and was free from protein contaminations as judged by immunoelectrophoresis. In SDS-PAGE under reducing and unreducing conditions PCI showed a single band at Mr = 57,000. The specific activity of the inhibitor was 226 units/mg. Surprisingly, the isolated PCI inhibited the amidolytic activity of urokinase (u-PA) on Glu-Gly-Arg-pNA (S-2444) in a time-dependent manner. Heparin, dextran sulfate and pentosanpolysulfate accelerated the reaction catalytically. PCI revealed itself as a non-competitive inhibitor of u-PA. The Ki-value was determined to be 7.9 x 10(-8)M. Inhibition of amidolytic activity was found to be associated with the formation of an 1:1 equimolar complex with a Mr of 110,000 as demonstrated by means of polyacrylamide gel electrophoresis and following Western blotting technique using polyclonal antibodies against u-PA and PCI. The specific activity of the isolated PCI of 226 units/mg, which approximates the theoretical value of pure PCI, indicates a highly purified preparation of PCI. The heparin-dependent inhibition of urokinase by this highly purified protein as well as comparison of the kinetic data and amino-acid composition of both PCI and the recently described plasminogen activator inhibitor (PAI) 3 give high evidence of identity of PCI and PAI-3.

Amino Acids↗
PubMed1987

[Properties and virus safety of a pasteurized antithrombin III concentrate].

In the last decade it appeared more than once that blood donations were contaminated by hitherto unknown viruses. Due to this fact, further steps have to be included into the manufacturing procedure of even up to now safe products from human plasma to eliminate potential new risks. A procedure is described which enables the pasteurization of antithrombin III analogous to albumin without major changes in the properties of the molecule. Thus the new product Kybernin HS/P was obtained. The efficacy of the pasteurization step was tested using relevant human pathogenic viruses. The following titers of important viruses could be totally inactivated within the pasteurization time (h) given in brackets HIV: (human immunodeficiency virus) greater than or equal to 10(6.7) (1), CMV (cytomegaly virus) greater than or equal to 10(4.5) (1), HSV (Herpes simplex virus) greater than or equal to 10(6.8) (4), Polio (poliomyelitis virus) greater than or equal to 10(6.9) (4).

Antithrombin III↗
PubMed1987

Virus safety of pasteurized factor VIII and factor IX concentrates: study in virgin patients.

In a long term surveillance study hemophilia A and B patients were treated with a Factor VIII HS and Factor IX HS concentrate respectively, both pasteurized by heating in solution: 10 hours at 60 degrees C. None of 31 virgin hemophiliacs treated with Factor VIII HS Behringwerke developed hepatitis B during a follow up between 6 to 60 months. One patient who received 379.280 IU Factor VIII by 977 applications showed a seroconversion after 961 days of treatment. Passive/active immunisation is suggested. 4 patients had moderate elevations of transaminases (less than 120 U/l) without clinical signs of liver disease. 2 patients suffered a non-icteric NA NB-hepatitis two months after synovectomy in the same hospital. 6 virgin hemophilia B patients who had been treated with Factor IX concentrate HS Behringwerke remained serologically negative and did not develop any symptoms indicative of a hepatic disease during a follow up between 11-29 months. The HTLV-III safety of Factor VIII HS and Factor IX HS Behringwerke is presently under investigation by determination of the corresponding antibody.

Adolescent↗
PubMed1986

New chromogenic substrates for thrombin with increased specificity.

Chromogenic substrates for thrombin with high specificity are necessary for several functional assays, especially for the performance of photometric PT and APTT. A new approach to improve the specificity of chromogenic peptide substrates is made coupling tripeptide sequences selective for thrombin to derivatives of 5-amino-2-nitro benzoic acid (ANBA). Especially when the chromophore's side chain is substituted by amines or amino acids hydrolysis rates by other enzymes like kallikrein, plasmin or factor Xa are decreased significantly compared to corresponding para-nitroanilides of the same amino acid sequence. On the other hand, most of these compounds are still sensitive thrombin substrates. KM-values for thrombin and other enzymes are in the same order of magnitude as corresponding pNA-peptides. ANBA peptide substrates may be useful to measure thrombin selectively in a mixture of other proteases like plasmin, factor Xa or kallikrein and for the colorimetric determination of PT and APTT.

Chromogenic Compounds↗
PubMed1986

Fibronectin decreases pulmonary vascular permeability under baseline conditions and after administration of arachidonic acid in rabbit lungs.

In blood- and plasma-free perfused isolated rabbit lungs, the influence of albumin and soluble fibronectin on vascular permeability was investigated. The lungs were perfused with Krebs Henseleit buffer containing no protein (KHB), containing 1 g/100 ml bovine albumin (KHAB) or containing albumin together with 100 micrograms/ml soluble fibronectin. The absence or presence of albumin had no influence on the perfusion pressure, the vascular compliance and the capillary filtration coefficient (CFC), determined by zero time extrapolation of the slope of weight gain which was induced by a sudden venous pressure elevation. Fibronectin caused a slight increase in pulmonary artery pressure, a slight decrease in vascular compliance and an approximately 50% reduction of CFC. In a second set of experiments, KHAB-perfused lungs were stimulated by the administration of 100 microM arachidonic acid (AA) during the second hydrostatic challenge within a sequence of three venous pressure elevations. In the presence of indomethacin, which blocks any significant increase in pulmonary vascular pressure after AA application, this procedure caused an immediate gain in lung weight due to increased pulmonary vascular permeability, with greater than 10-fold increased CFC values subsequent to the AA application. In the presence of 100 micrograms/ml fibronectin, this AA-induced increase in CFC was mitigated to less than 20% of the controls without the glycoprotein, with correspondingly severalfold reduced lung weight gain. In conclusion, the present study provides evidence for a direct influence of circulating soluble fibronectin on lung microvascular integrity and fluid balance under baseline conditions and after stimulation of the pulmonary AA cascade.

Animals↗
PubMed1986

Study on the mechanism of action of heparin and related substances on the fibrinolytic system: relationship between plasminogen activators and heparin.

It is now generally well accepted that heparin and related substances increase the fibrinolytic activity in vivo. The stimulation of the amidolytic, plasminogenolytic and fibrinogenolytic activity of tissue plasminogen activator and urinary plasminogen activator through heparin was investigated in vitro. A concentration-dependent stimulation of the plasminogenolytic and fibrinogenolytic activity of both urinary and tissue-type plasminogen activators was observed in the presence of heparin. No heparin dependence was observed in the amidolytic assay. Heparin stimulates the plasminogenolytic activity of tissue plasminogen activators in the same manner as fibrin. Both activators form complexes with heparin; the heparin-binding-site seems to be identical or related with the fibrin-binding-site of tissue plasminogen activator. The physiological role of these interactions is discussed.

Chromatography, Affinity↗
PubMed1986

Physicochemical, immunochemical and functional comparison of human S-protein and vitronectin. Evidence for the identity of both plasma proteins.

The comparison of the complement inhibitor s-protein, isolated from human plasma, with vitronectin, a serum spreading factor, revealed a high degree of similarity of both proteins with respect to molecular weight, band pattern in polyacrylamide gels in the presence of sodium dodecyl sulfate and amino acid composition. While radiolabeled S-protein was precipitated by antiserum against vitronectin, both proteins exhibited precipitin lines of complete identity in double immunodiffusion analysis when tested mutually against antisera of the appropriate components. The functional property of vitronectin to promote cell spreading of fibroblasts was also documented for purified S-protein. These findings indicate a high degree of similarity with respect to structural and functional properties of S-protein and vitronectin and hence may implicate that both proteins are identical.

Cell Adhesion↗
PubMed1986

[Methodological aspects of the photometric determination of prothrombin time using chromogenic substrates].

A method for photometric determination of prothrombin time (PT) with a chromogenic peptide as substrate is described. The reagent contains human placental thromboplastin, a chromogenic substrate, calcium, a heparin antagonist and buffer. The new prothrombin time method has been calibrated against international reference preparations for thromboplastin. The reagent is sensitive to deficiency of all coagulation factors of the extrinsic pathway. However, it is not sensitive for heparin up to 1 IU/ml. The precision of this fast and simple method is comparable to that of mechanised assays for clinical chemistry.

Blood Coagulation↗
PubMed1985

Histidine-rich glycoprotein and changes in the components of the fibrinolytic system after streptokinase therapy in patients with pulmonary thromboembolism.

Although the biological function of histidine-rich glycoprotein (HRG) is unknown, it may serve as an antifibrinolytic agent by interfering with the binding of plasminogen to fibrin. To define the role of HRG, plasma titers were measured by single radial immunodiffusion in eleven patients with thromboembolism before and after streptokinase (SK) therapy and were found unchanged (84.7 +/- 6.2%, M +/- SEM before, and 99.5 +/- 6.3% after 12 hr of SK therapy). The HRG peaks on crossed immunoelectrophoresis before and after SK infusion were also unchanged. Alpha 2-plasmin inhibitor fell during SK infusion as measured immunologically (102.0 +/- 15.0% before and 28.0 +/- 1.6% after 12 hr of therapy) and fibrinogen-fibrin degradative products appeared (mean titer of 1:2,048 after 12 hr of therapy), indicating that the infused SK was biologically active. Plasminogen levels before therapy were normal, as measured functionally and immunologically (105.4 +/- 4.9% and 96.0 +/- 5.6%, respectively), and both decreased after 12 hr of SK therapy (15.2 +/- 5.6% and 50.8 +/- 4.3%). No changes in functional antithrombin III titer, Hageman factor antigen level, or fibrinogen concentration, as measured turbidimetrically, were observed. Thus, although these data do not allow one to make any firm conclusions regarding the physiologic role of this protein in fibrinolysis, they do not exclude its increased catabolism, compensated by increased production, in patients undergoing fibrinolytic therapy.

Blood Proteins↗
PubMed1985

Discrimination between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide.

Circulating soluble fibrin, observed in the blood of patients with ongoing intravascular coagulation, is generated from the plasma protein fibrinogen by the limited proteolytic action of thrombin. We report the production of a monoclonal antibody that discriminates between fibrin and fibrinogen in blood. The synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the alpha chain of human fibrin, was used as immunogen. This hexapeptide is located within the A alpha chain of fibrinogen but becomes the amino terminus of the fibrin alpha chain, after fibrinopeptide A is removed by the action of thrombin, and thus becomes accessible for antibody binding. The monoclonal antibody we have prepared can discriminate between fibrin and fibrinogen and thus can be used in assay systems to quantitate soluble fibrin or, potentially, to image fibrin-rich thrombi.

Amino Acid Sequence↗
PubMed1985

Acquired thrombin inhibitor in a patient with liver cirrhosis.

An unusual thrombin inhibitor is described in a patient suffering from liver cirrhosis, associated with a polyclonal gammopathy. In all screening tests that are based on thrombin action, the plasma clotting time was prolonged; in contrast, a test using the thrombin-like enzyme thrombin coagulase was not influenced. A specific thrombin inhibitor, present in the patient's plasma, was assumed. Fractionation experiments showed that the inhibitor was associated with the patient's IgG fraction. The IgG fraction dose-dependently inhibited the action of thrombin on fibrinogen and the inhibition of thrombin by antithrombin III, but the amidolytic activity of thrombin was only little affected.

Aged↗
PubMed1982

Reduced histidine-rich glycoprotein levels in plasma of patients with advanced liver cirrhosis. Possible implications for enhanced fibrinolysis.

Histidine-rich glycoprotein is a 3.8s alpha 2-glycoprotein of human plasma originally isolated in 1972 [1,2]. The biologic function of histidine-rich glycoprotein, however, is unknown. A recent report suggests that histidine-rich glycoprotein binds to the high-affinity lysine-binding sites of plasminogen and that histidine-rich glycoprotein may retard fibrinolysis by interfering with the binding of plasminogen to fibrin [3]. We have measured the plasma titers of histidine-rich glycoprotein in normal subjects and patients with advanced hepatic cirrhosis by single radial immunodiffusion with a monospecific antiserum. The levels in 22 patients were 7.0 +/- 2.5 mg/dl (mean +/- SD), whereas those in 20 control subjects were 11.8 +/- 2.7 (p less than 0.001). Upon two-dimensional crossed immunoelectrophoresis, the pattern of histidine-rich glycoprotein in liver cirrhosis was similar to that of normal histidine-rich glycoprotein. Since histidine-rich glycoprotein seems to function as an antifibrinolytic agent, the decreased titers in cirrhosis may be one factor contributing to the enhanced fibrinolysis commonly seen in this disorder.

Adult↗