PubMed Health⌕ Search

Biomedical subjects

F Brosstad

Publications and source records attributed to F Brosstad.

At least 73 records · Page 4Linked to original sources
PubMed1995

Roentgen contrast media and fibrinolysis.

A review of the literature on the influence of contrast media (CM) on fibrinolysis showed that information currently available is contradictory, inconclusive and fragmentary. Results of some in vivo studies suggest that both ionic and nonionic CM increase fibrinolytic activity, either by lowering plasma plasminogen activator inhibitor 1 levels, by releasing endogenous tissue plasminogen activator and¿or by altering the fibrin meshwork of thrombi. Most in vitro studies on the subject contain results that are contradictory to those from in vivo studies. The discrepancies are due to obvious differences between systems which are, at best, only comparable under conditions of stagnant flow. Thus, great care should be exercised when extrapolating or interchanging data obtained in vivo and in vitro.

Animals↗
PubMed1995

Stimulated Glanzmann's thrombasthenia platelets produced microvesicles. Microvesiculation correlates better to exposure of procoagulant surface than to activation of GPIIb-IIIa.

The mechanism of formation of platelet-derived microvesicles remains controversial. The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann's thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 microM), and the thrombin receptor agonist peptide SFLLRN (100 microM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 microM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 +/- 2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 +/- 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 microM SFLLRN gave 16.3 +/- 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 +/- 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.

Adult↗
PubMed1994

Fibrinogen present in EDTA--anticoagulated plasma stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

The presence of soluble fibrin in plasma is an early and sensitive indicator of activation of the coagulation system. Quantitative spectrophotometric assays for soluble fibrin can be based on the principle that soluble fibrin stimulates the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. It was previously shown that treatment of purified fibrinogen by EDTA, which removes the three tightly bound Ca2+ ions, results in exposure of tissue-type plasminogen activator-catalytic sites similar to those unveiled by thrombin. Since EDTA is a common anticoagulant, it was of interest to study the effect of EDTA on a test based on plasminogen activation. It is concluded that the determination of soluble fibrin in EDTA-anticoagulated plasma from healthy individuals gives a false positive indication of the presence of soluble fibrin. This was true irrespective of whether the test was performed at pH 7.4, 7.8 or 8.5. The most probable explanation is that tissue-type plasminogen activator-stimulating sites are exposed in fibrinogen by EDTA. Therefore, EDTA-plasma is unsuitable for assaying soluble fibrin with tests based on the tissue-type plasminogen activator-mediated conversion of plasminogen to plasmin.

Amino Acid Sequence↗
PubMed1994

The effect of various contrast media on the activation of plasminogen by streptokinase or recombinant tissue plasminogen activator in vitro.

RATIONALE AND OBJECTIVES: Radiologic contrast media (CM) may influence processes of coagulation and fibrinolysis. In the current study, the effects of various CM on the formation of plasmin were examined in an in vitro buffer system. METHODS: The effects of three clinically relevant concentrations of seven different iodine-containing CM and gadolinium-DTPA on streptokinase (SK) or recombinant tissue plasminogen activator (rt-PA)-induced plasmin formation was monitored using a plasmin-sensitive chromogenic substrate. RESULTS: Contrast media generally had an inhibitory effect at the plasminogen activation step; this effect was particularly noticeable with the ionic CM. CONCLUSIONS: Contrast media influence plasminogen activation by SK and rt-PA in vitro. Ionic CM have a more pronounced inhibitory effect than the nonionic media. The ionic Gd-DTPA shows a less inhibitory effect than the ionic iodine-containing CM. However, they must be regarded separately because of the different chemical composition of the magnetic resonance imaging and x-ray CM.

Contrast Media↗
PubMed1994

Freeze-dried fibrinogen or fibrinogen in EDTA stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin.

Both soluble and insoluble fibrin stimulate the tissue-type plasminogen activator-catalysed conversion of plasminogen to plasmin. Whether fibrinogen can exert a similar effect has been a controversial issue. The present investigation shows that while fibrinogen purified by beta-alanine precipitation does not stimulate the tissue-type plasminogen activator-catalysed plasminogen activation, fibrinogen which has been either lyophilized or stripped of bound Ca2+ ions by EDTA chelation, stimulates this reaction. The data indicate that such procedures alter the molecular conformation of fibrinogen, and expose stimulatory sites which are hidden in the native fibrinogen molecule. These results may explain previous findings concerning the capacity of fibrinogen as a stimulator of the tissue-type plasminogen activator-catalysed plasminogen activation. Since even slight alteration of the molecular structure of fibrinogen leads to an increase in the tissue-type plasminogen activator stimulation, the authors suggest that this can be used to test if the fibrinogen is in a native state.

Amino Acid Sequence↗
PubMed1994

Demonstration of platelet-derived microvesicles in blood from patients with activated coagulation and fibrinolysis using a filtration technique and western blotting.

Platelet vesiculation in vitro is correlated to platelet activation. It was therefore considered of interest to see if microvesicles (MV) are present in blood in clinical situations associated with platelet activation in vivo. Patients with both activated coagulation and fibrinolysis, implying that thrombin has been generated, suit such a purpose. Thus, the aim of this study was to investigate whether microvesicles could be detected in patients with activated coagulation and fibrinolysis, as diagnosed by the presence of soluble fibrin (positive ethanol gelation tests) and positive tests for fibrin degradation products (FDP). Platelet-rich plasma was prepared from citrated blood from patients (n = 22) and healthy controls (n = 32) matched as to age and sex. The intact platelets were removed from plasma by centrifugation. Any MV present were isolated from the platelet-free plasma by a filtration procedure, washed, solubilized in Triton X-100 and subjected to SDS-PAGE with Western blotting using a MAb against GPIIb alpha as an indicator of the presence of microvesicles. All of the 22 patients showed the presence of microvesicles detectable by the content of GPIIb alpha, whereas this could be observed in only 4 out of the 32 normal controls and then in small or trace amounts only. The presence of microvesicles among cell-derived material in the plasma of two of the patients was also confirmed by electron microscopy. To the best of our knowledge this is the first report on the presence of microvesicles in plasma from patients with both activated coagulation and fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Comparison of three D-dimer assays for the diagnosis of DVT:ELISA, latex and an immunofiltration assay (NycoCard D-Dimer).

Ninety-two consecutive patients referred for suspicion of deep venous thrombosis (DVT) were analyzed for D-dimer using ELISA, latex test, and a new immunofiltration method (NycoCard D-Dimer). Contrast venography verified the diagnosis in 40, and excluded the diagnosis in 52 patients. The sensitivity, negative predictive values, specificity and positive predictive values were, for ELISA 98%, 95%, 38% and 54, for NycoCard D-Dimer 100%, 100%, 42% and 57% and for the latex test 73%, 78%, 75%, and 69%, respectively. Sensitivity and specificity were inversely related with increasing pathological cut-off value. Comparison of test results by concentration category revealed a good agreement between ELISA and NycoCard D-Dimer, but to less extent between latex and the two other tests. It is concluded that NycoCard D-Dimer and D-dimer ELISA are well-suited as exclusion tests for DVT. A plasma sample is tested with NycoCard D-Dimer in less than 2 min. Thus, this test combines advantageous analytical properties comparable to the ELISA-test, with rapidity and simplicity comparable to the latex test.

Adult↗
PubMed1993

The difference between platelet and plasma FXIII used to study the mechanism of platelet microvesicle formation.

The formation of microvesicles from platelets was induced either by activation of the complement system by a monoclonal antibody to CD9, or by incubation of platelets with the calcium ionophore A23187. A filter technique to isolate the microvesicles without plasma contamination is described. The microvesicles contained FXIIIa2 from the platelet cytoplasm which shows that these particles contain significant amounts of intracellular material. This was shown by the use of crossed immunoelectrophoresis with rabbit antibodies to total human platelet proteins in the second dimension gel and polyclonal antibodies against the a- and b-subunit of FXIII in the intermediate gel. The FXIIIa2 in the microvesicle was found to be functional as an enzyme. To prove this, it was shown that FXIII in its immunoprecipitate arc could catalyze the incorporation of monodansylcadaverine into casein as identified by fluorescence of this arc in ultraviolet light. The observation that the plasma form of FXIII (FXIIIa2b2) was absent from the microvesicles collected by the filtration technique, whereas it was present in platelet fragments obtained by mechanical disruption by ultrasonication, indicates that the activation-dependent microvesicles are formed by a true budding process with the inclusion of intracellular, but not extracellular material.

Antibodies, Monoclonal↗
PubMed1993

[Von Willebrand's disease--gene, molecule, clinical findings, treatment].

In 1926, Erich von Willebrand described a bleeding disorder which, because of its autosomal inheritance and the tendency for spontaneous mucocutaneous bleeding, he called hereditary pseudohaemophilia. As a tribute the disease was named after him. However, its etiology and pathogenesis remained obscure until 1971, when Zimmermann and co-workers discovered that the blood of von Willebrand patients contained a lower than normal level of an antigen, the von Willebrand factor, which was associated with coagulation factor VIII. This paper briefly reviews the main points of what is known today about von Willebrand's disease: subtypes, gene, von Willebrand factor molecular/biological function relationships, prevalence, clinical expression, diagnosis and treatment.

Diagnosis, Differential↗
PubMed1993

[Diagnosis of hemoglobinopathies and thalassemias].

During recents the number of immigrants to Norway from Africa and South East Asia has risen considerably. These persons come from countries with a high prevalence of haemoglobinopathies. Most of the immigrants with haemoglobinopathies are silent carriers, but some have a serious disease or have offspring with serious disease. This situation calls for increasing awareness and knowledge of haemoglobinopathies, especially the thalassemias and sickle cell trait. Genetic counselling is particularly important, since the majority of these immigrants marry within small ethnic groups. This paper gives a survey of the most frequent haemoglobinopathies likely to be encountered in Norway.

Africa↗
PubMed1993

Cross-linked alpha s gamma t-chain hybrids in plasma clots studied by 1D- and 2D electrophoresis and western blotting.

Fibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the alpha-chains or the gamma-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute alpha gamma-chain hybrids or superimposed alpha- and gamma-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia's Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains alpha s gamma t-chain hybrids and gamma-polymers, in addition to the well-known gamma-dimers and alpha-polymers. The main alpha s gamma t-chain hybrid has a pI between that of the alpha- and the gamma-chains, a MW of about 200 kDa and contains A alpha-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded alpha-dimers with MWs close to the gamma-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

Blotting, Western↗
PubMed1993

Characterization of fibrinogen/fibrin derivatives isolated from normal and fibrinaemic plasma and serum using paramagnetic particles coated with a monoclonal antibody (mAb) to D-dimer.

Paramagnetic particles coated with a monoclonal antibody to D-dimer (mAb S4) were used to isolate and concentrate D-dimer and D-dimer-containing complexes in plasma and serum. Antibody-captured material was eluted with SDS-urea buffer and examined by either SDS-polyacrylamide or submerged SDS-agarose gel electrophoresis followed by Western blotting. The protein pattern was visualized by either polyclonal antibodies to human fibrinogen or monoclonal antibodies specific for fibrinogen derivatives containing fibrinopeptide A (FpA; mAb Y18), the N-terminus of the beta-chain in fibrin (mAb 59D8) or the gamma-chains (mAb J88B). The results obtained show that paramagnetic particles coated with mAb S4 catch intact D-dimer as well as a variety of cross-linked fibrin molecules of high-molecular-weight (HMW) in plasma. The existence of HMW derivatives in fibrinaemic serum indicates that some of the HMW fibrin related material in such plasma is not clottable. Fibrinogen/fibrin monomers and some of the fibrinogen/fibrin related derivatives found in the eluates were probably non-covalently bound to the complexes caught by mAb S4 coated particles. The present technique combines the selective concentrating power of immunoparticles and the sensitivity of immunovisualization and allows rapid and direct identification of minute amounts of circulating immunoreactive fibrinogen/fibrin derivatives.

Antibodies, Monoclonal↗
PubMed1993

A rapid method for selecting specific hybridoma clones using paramagnetic Dynabeads.

This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X-63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti-mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD. Mixing such beads with the fused cells allowed selective harvesting of cells with membrane-expressed anti-DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96-well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40-79% of the tested clones. It is concluded that it is possible to use antigen-coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest.

Animals↗
PubMed1993

Platelets in shed mediastinal blood used for postoperative autotransfusion.

Ten patients undergoing open-heart surgery received postoperative autotransfusion of shed mediastinal blood collected in the cardiotomy reservoir. The number, function and morphology of the platelets found in the shed blood were investigated. Platelets were counted using an electronic counter compared with light microscopy. Morphology of platelets was studied with electron microscopy. Platelet aggregation was studied using an aggregometer. Dense granule secretion was measured as the extracellular appearance of adenosine triphosphate. Enumeration of platelets in shed blood using the two methods gave different results. Thus, the electronic counter gave a mean platelet count of 62 x 10(9).l-1, while light microscopy revealed only a mean platelet count of 10 x 10(9).l-1. Electron microscopy disclosed few platelets, but numerous cytoplasmatic fragments smaller than or up to the same size as platelets. The platelets found were mostly shape-changed, spheroid, characterized by centralization and loss of alpha-granules and dense bodies, all changes that indicated irreversible platelet activation. The platelets failed to aggregate in response to the presence of thrombin, adenosine diphosphate or collagen, and secretion of adenosine triphosphate was absent. Plasma from the shed blood was not capable of inducing spontaneous aggregation in platelet-rich plasma from healthy donors. These results indicate that infusion of larger volumes of autotransfused blood should be supplemented with platelet concentrates.

Adenosine Diphosphate↗
PubMed1993

Assay of D-dimer based on immunofiltration and staining with gold colloids.

In this immunofiltration assay of D-dimer in plasma samples, the antigens are captured by a monoclonal antibody on a porous membrane, and labeled with the same antibody conjugated to gold colloids. The assay time is < 2 min, and a color of intensity proportional to the concentration of D-dimer is left on the membrane. The reference range (mean +/- 2 SD) was 0.336 +/- 0.133 mg/L (n = 69). Linearity was found up to 10 mg/L. Comparison with ELISA results (x) for 198 patients' samples demonstrated a linear regression equation of y = 0.99(+/- 0.05)x + 0.68(+/- 0.07) and a mean square error of 0.503. Comparison of visual reading of the color signal (y) vs reflectometric measurements (x) for 220 patients' samples demonstrated a linear regression equation of y = 2.5(+/- 0.06)x -0.22(+/- 0.04) and a mean square error of 0.095. Bilirubin, hemoglobin, fibrinogen, soluble fibrin, and fibrinogen degradation products and freezing/thawing of samples did not interfere. Some interference from rheumatoid factor, heparin, and the presence of cells or large lipid particles was seen. The variance (CV) was 8-12% within run, 10-18% between runs, and 13-20% between persons. The new assay constitutes a rapid and reliable analytical tool combining simplicity equivalent to that of latex tests with analytical information approaching that of ELISA.

Drug Stability↗
PubMed1992

Soluble, cross-linked fibrin(ogen) hybrid oligomers do not stimulate t-PA conversion of plasminogen.

Cross-linked hybrid oligomers of fibrinogen and fibrin are found in plasma from fibrinaemic patients and normal individuals as well as in preparations of purified human fibrinogen. The present study was undertaken to see if such hybrid oligomers have the same stimulatory effect on tissue plasminogen activator (t-PA) conversion of plasminogen as do polymeric and monomeric fibrin. Hybrid oligomeric fibrin(ogen) material was provided by subjecting purified human fibrinogen to gel filtration in urea-containing buffer at pH 5.6. Well separated fractions of hybrid oligomeric material and monomeric fibrinogen were thus obtained. Some of this material was converted to soluble polymeric or monomeric fibrin using insolubilized thrombin. Hybrid polymeric fibrin, polymeric fibrin or monomeric fibrin were then added to citrated, normal plasma to 2.5 or 5 per cent of the plasma fibrinogen concentration. The added material was kept in solution by plasma fibrinogen. The "COA-SET Fibrin Monomer Test" (Kabi,Stocholm,Sweden), based on the ability of fibrin monomers to enhance t-PA mediated plasminogen-plasmin conversion, was used to compare the potential stimulatory effect of the preparations above. The results led to the following conclusions: 1) Cross-linked, soluble fibrin(ogen) hybrid polymers in a concentration of 5 per cent of plasma fibrinogen concentration (w/w) do not stimulate t-PA. 2) Thrombin conversion of the fibrin-fibrinogen hybrid material resulted in an increase in the rate of t-PA mediated plasminogen conversion, corresponding to the one observed with equivalent (w/w) amounts of fibrin monomers. Compared on a mole to mole basis, fibrin oligomers are more powerful than fibrin monomers as stimulators of t-PA activity.

Chromatography, Gel↗