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Biomedical subjects

F Brosstad

Publications and source records attributed to F Brosstad.

At least 91 records · Page 5Linked to original sources
PubMed1992

Early cross-linked fibrin in human plasma contains alpha-polymers with intact fibrinopeptide A.

alpha-polymer formation, as opposed to gamma-chain dimerization has been considered a relatively late event in factor XIII-induced fibrin stabilization. Recently it has been shown, however, that plasma from healthy individuals and from patients with fibrinaemia contains small amounts of soluble fibrin/fibrinogen oligomers interlinked through dimerized gamma-chains as well as cross-linked alpha-chains. The present work was carried out to see if these early alpha-chain polymers also arise during coagulation of plasma in vitro. Plasma samples from healthy individuals, prepared by immediate centrifugation of blood collected without anticoagulant, were allowed to clot spontaneously for varying periods. The plasma clots were solubilized in SDS-urea-mercaptoethanol and samples were subjected to SDS-PAGE and Western blotting using polyclonal antibodies to human fibrinogen, or monoclonal antibodies specific either for A alpha/alpha-chains, for fibrinopeptide A-containing chains, for the N-terminus of the fibrin beta-chain or for the gamma-chains. Fibrin/fibrinogen oligomers were seen to form long before visible gelation of plasma. These oligomers were cross-linked through gamma-chain dimerization, but also through A alpha- or alpha-chain polymerization. The number and amount of alpha-polymers containing A alpha-chains increased immediately after clot formation, but these disappeared about 20 min later, due to complete removal of fibrinopeptide A (FPA) by thrombin. It is concluded that alpha-polymer formation is a very early event during plasma coagulation in vitro, and that both A alpha- and alpha-chains are involved.

Blood Coagulation↗
PubMed1992

Membrane fluidity and platelet aggregation: dibucaine permits ristocetin-induced platelet aggregation with low-molecular-weight von Willebrand multimers.

The effects of brief incubation of platelets with dibucaine were studied. Membrane fluidity was increased after incubation with 1 mM dibucaine for 1 min as determined by fluorescence polarization. Platelet aggregation was increased when ristocetin was employed as modulator and normal plasma used as a source of von Willebrand factor (vWF). In contrast, aggregation was decreased with botrocetin. After cryoprecipitation the supernatant, which contained predominantly low-molecular-weight vWF multimers, was effective with ristocetin only after prior incubation of the platelets with dibucaine. This indicates that low-molecular-weight vWF multimers can also support ristocetin-induced aggregation when the membrane fluidity is increased. This idea was supported by the use of plasma from a patient with von Willebrand disease type IIa. Being a multimeric protein, von Willebrand factor contains multiple binding sites for glycoprotein Ib (GP Ib). GP Ib-dependent aggregation by agents that do not contain multiple binding sites, i.e. wheat germ agglutinin or polyclonal antibodies to GP Ib, was decreased after brief incubation with dibucaine. These observations are discussed in relation to the hypothesis that the increased membrane fluidity allows an increased number of GP Ib molecules to bind to each vWF multimer.

Crotalid Venoms↗
PubMed1991

Hybridomas can successfully be prepared from frozen/thawn spleen cells.

Spleens from mice immunized with either human von Willebrand factor, albumin or fragment DD from plasminolyzed fibrin were divided into two equal parts. Subsequently, one half of the spleen cells were fused directly with X-63 mouse myeloma cells and the other half fused after freezing and thawing. The results show that hybridomas may successfully be prepared from frozen/thawn spleen cells, but that a higher cell density than with fresh cells is required. The possibility of making use of frozen spleen cell material implies that valuable time and cell material can be saved, and that fusions can be postponed and performed at the time of choice.

Animals↗
PubMed1991

Gastric lymphoma causing granulocytopenia and cold intolerance, with recovery after treatment.

A 34-year-old man was found to have granulocytopenia with a white blood count of 2.3 x 10(9) l-1, consisting of 10% segmented neutrophils, 50% monocytes and 40% lymphocytes. A bone marrow aspirate showed 20% promyelocytes and 10% blasts with monoblastic features, and a smouldering myelomonocytic leukaemia was considered to be a possible diagnosis. In cold weather the patient experienced cold intolerance with acrocyanosis and small ulcerations on the ears. The test for heparin-precipitable protein ('cryofibrinogen') was strongly positive. During the following year, these signs and symptoms persisted, and the patient also developed constant moderate pain in the epigastric region. Gastroscopy revealed a large lymphoma of the stomach, which was a high-grade malignant centroblastic type of non-Hodgkin's lymphoma. After successful removal of the tumour, and six courses of potent cytostatic combinations, the patient recovered completely, and the granulocytopenia and cold intolerance disappeared.

Adult↗
PubMed1991

Autotransfusion after open heart surgery: characteristics of shed mediastinal blood and its influence on the plasma proteases in circulating blood.

Fourteen patients undergoing open-heart surgery received intermittent or continuous postoperative autotransfusion of shed mediastinal blood (minimum 400 ml during 6 h after surgery) collected in the cardiotomy reservoir. Hematologic variables and changes in the coagulation, fibrinolytic and plasma kallikrein-kinin systems were investigated in the reservoir blood at the beginning and after 6 h of autotransfusion, and in patient blood during and after surgery and before and after autotransfusion. Autotransfusion volume ranged from 400 to 1200 ml per patient (median 482 ml). The reservoir blood had a median haemoglobin level of 93 and 74 g/l, a platelet count of 71 and 119 x 10(9)/l, and plasma haemoglobin level of 3110 and 4100 mg/l before and after 6 h of autotransfusion, respectively. Further examination of the reservoir blood showed that it had undergone extensive coagulation and fibrinolysis as well as a moderate activation of the kallikrein-kinin system. Despite these extensive alterations in the reservoir blood, no major change could be found in the circulating blood after autotransfusion, except for a moderate increase in plasma haemoglobin from 180 mg/l to 430 mg/l. The clinical safety and simplicity of this technique were confirmed for autotransfusion of shed mediastinal blood up to 1200 ml.

Aged↗
PubMed1991

Characterization of a human trypsin resistant to Kunitz soybean trypsin inhibitor. Studies of duodenal juices after tube instillation of raw soybean extract.

Human duodenal juices collected during tube instillation of raw soybean extract into the duodenum contained free trypsin and free Kuntiz soybean trypsin inhibitor (KTI) in the simultaneous presence of trypsin-KTI complexes. It has previously been suggested that this KTI-non-inhibitable trypsin has a general resistance to serine protease inhibitors. Four different trypsin forms have been found and partly characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing followed by Western immunoblotting or enzyme staining. In addition, crossed immunoelectrophoresis and affinity chromatography with antibody-coupled gels have been used for identification of free and inhibitor-complexed trypsin.

Blotting, Western↗
PubMed1990

[Are prosecuted parents allowed the benefit of the doubt in cases of child abuse?].

The justified campaign against child abuse has unfortunately had a side effect. It has ruined the lives of some innocent parents of children with undiagnosed osteogenesis imperfecta. For 15 years, Colin Paterson and co-workers have studied a large number of patients with type IV of osteogenesis imperfecta, and have found that more than 50 per cent of them have normal radiographs of the bones at the time of the first fracture. Paterson and co-workers have also found that fractures of the ribs and skull are by no means uncommon in osteogenesis imperfecta type IV. These important observations should help, in the future, to prevent prosecution of innocent parents of children with osteogenesis imperfecta type IV, provided that the observations are not overlooked by pediatricians.

Child↗
PubMed1990

Normal and fibrinaemic patient plasma contain high-molecular weight crosslinked fibrin(ogen) derivatives with intact fibrinopeptide A.

Freshly drawn plasma samples from healthy subjects and from fibrinaemic patients were subjected to electrophoresis on SDS-agarose (unreduced material) or on SDS-PAG (1D and 2D, reduced material) and Westernblotted. The blots were immunovisualized using either polyclonal anti-fibrinogen or a monoclonal antibody (Y18) to fibrinopeptide A-containing molecules. The following results were obtained: 1. Normal plasma as well as plasma from patients with fibrinaemia contained FXIII-crosslinked HMW oligomers, stabilized through gamma gamma-dimerization as well as alpha-polymer formation and these oligomers contained molecules with intact A alpha-chain N-termini. 2. Cross-linked material amounted to less than 0.1% of the fibrinogen pool regardless of the sample studied, and apparently less in fibrinaemic patient plasma than in normal plasma. Thus, since the ratio of crosslinked fibrin(ogen) derivatives to that of fibrinogen was lower for fibrinaemic plasma than for normal plasma, it is suggested that the type of soluble fibrin which gives rise to a positive EGT in fibrinaemia patients is not crosslinked.

Adolescent↗
PubMed1990

Is the RPM cell line a useful model for the study of megakaryocyte development?

The differentiation of megakaryoblasts into megakaryocytes and their release of blood platelets are complex and poorly understood processes. As an aid to investigate this process several cell lines with megakaryocyte characteristics have been established. One of these cell lines is the rat promegakaryoblast-like (RPM) cell line established by Cicoria and Hempling and used by others to describe maturation processes in megakaryocytes. We have used this cell line to study the synthesis of platelet-specific marker proteins. Severe difficulties led us to perform control experiments to confirm earlier findings. We were unable to confirm several of the previous reports, and we conclude that this particular cell line should not be recommended for the study of megakaryoblast differentiation.

Acetylcholinesterase↗
PubMed1988

Immunovisualization of fibrinogen A alpha-chain heterogeneity in normal plasma and plasma from patients with DIC or on streptokinase therapy.

Purified fibrinogen as well as normal plasma, or plasma from patients with DIC or undergoing streptokinase(SK)-therapy was subjected to 1D- and 2D SDS-electrophoresis under reducing conditions. The pattern was revealed either by Coomassie-staining or immunostaining after Western-blotting and then compared. The use of polyclonal antibodies to fibrinogen as well as two monoclonal anti-bodies reacting with FPA and C-terminal part of the A alpha-chain confirmed immunologically the previously reported molecular weight heterogeneity of the A alpha-chain of the fibrinogen molecule as being a constituent of normal plasma, and lead to the following conclusions: 1. The MW-heterogeneity is observed in the fibrinogen pool of normal plasma as well as in DIC-plasma, SK-plasma and in purified fibrinogen, being the least noticeable in normal plasma and most advanced in SK-plasma. Patterns obtained using immunostaining with monoclonal anti-FPA confirm that the MW-heterogeneity of fibrinogen is mainly due to C-terminal degradation of the A alpha-chain. 2. Numerous A alpha-chain remnants (at least 9), with intact N-terminal ends, are found to be present in normal plasma, with a MW range from 66,200 to 36,000 Da, demonstrating that each of the "classical" HMW, LMW, LMW' subgroups consist of fibrinogen molecules which are very heterogeneous. 3. Two populations of A alpha-chains in purified fibrinogen and in fibrinogen in plasma react with the C-terminal specific Mab G-8. This is in contrast to the findings in plasma from streptokinase-treated patients, where several bands of lower molecular weights than the gamma-chain can be seen, suggesting the presence of free, circulating A-alpha chains split in the N-terminal half of the chain beyond the last inter-chain disulphide bond. 4. 2D electrophoresis disclosed substantial deviations in the patterns obtained with DIC-plasma, SK-plasma and with fibrinogen purified by beta-alanine-precipitation from that observed with normal plasma. The present technique allows selective characterization of fibrinogen independently of the other proteins present in plasma and offers extreme sensitivity.

Blotting, Western↗
PubMed1988

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes.

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.

Animals↗
PubMed1987

Interactions of human blood platelets with three circulating plasma fibrinogens of different molecular weights.

The ability of three naturally occurring human fibrinogen species, HMW, LMW and LMW', to support ADP-induced platelet aggregation was investigated and compared to their ability to bind to gel-filtered platelets. Whereas HMW had intact subunit chains, LMW and LMW' had defined lesions in the C-terminal part of one (LMW) or both (LMW') of the A alpha-chains. The ADP-induced aggregation of gel-filtered platelets in the presence of LMW was about 75 per cent of that obtained with HMW (0.2 mumol/l of fibrinogen and 10 mumol/l of ADP). A mixture of equal amounts of LMW and LMW' showed around 50% decrease in aggregation. Compared to these difference in aggregation co-factor function, direct binding to gel-filtered platelets was less affected by the A alpha-chain degradation. However, a difference between LMW, LMW' and HMW binding was significant when the fibrinogens labelled with two different isotopes of iodine were present simultaneously. These results demonstrate that naturally occurring plasma fibrinogens differ in their interactions with platelets. As the HMW/LMW ratio changes during the acute phase, this may be of physiological significance.

Adenosine Diphosphate↗
PubMed1987

The N-DSK gamma-chain binds to immunoprecipitated GP IIb-IIIa.

The CNBr-split N-terminal disulphide knot of the fibrinogen molecule (N-DSK) binds to ADP-stimulated gel-filtered platelets and immunoprecipitated fibrinogen receptor. To investigate which part of the N-DSK molecule that is involved in this binding, the glycoprotein IIb-IIIa complex (the fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100 extracts of platelets against rabbit antibodies to whole platelet proteins. The immunoelectrophoresis plates were incubated with solubilized, carboxymethylated 125I-labelled A alpha -, B beta - or gamma-chains of N-DSK, and investigated for binding by autoradiography. The N-DSK gamma-chain, but not the A alpha - or B beta -chains demonstrated binding to the GP IIb-IIIa complex. These results show that the fibrinogen molecule contains a third sequence of amino acids, in addition to the two previously reported ones that can be involved in binding of fibrinogen to the fibrinogen receptor on the platelets.

Binding Sites↗
PubMed1987

Identification of platelet antigens by monoclonal antibodies using crossed immunoelectrophoresis with immunoblotting of the monoclonal antibody.

A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1986

Competitions between fibrinogen with its degradation products for interactions with the platelet-fibrinogen receptor.

Direct binding of 125-I-labelled plasmic and CNBr-derived fibrin (ogen) fragments (pre-X, X, Y, D, Degta, Efg, E1, N-DSK, N-dsk) to gel-filtered platelets was compared to their ability to support or inhibit ADP-induced aggregation, and to compete with fibrinogen for binding to ADP-stimulated platelets. Pre-X was the only fragment that supported aggregation. All fragments tested except for E derived from fibrinogen (Efg) and Degta bound specifically to the platelets and inhibited ADP-induced aggregation in the presence of fibrinogen. Competitive binding studies with fibrinogen and fragments labelled with different isotopes of iodine, or inhibition of binding of labelled fibrinogen with unlabelled fragments showed that all of the fragments except Efg and Degta were able to compete with fibrinogen for binding. When simultaneous binding of N-dsk and fibrinogen was studied, an increased binding of both ligands was observed probably due to complex formation. The results fully agree with previous findings of binding to immunoprecipitated glycoprotein IIb-IIIa after crossed immunoelectrophoresis. We conclude that the fibrinogen molecule contains at least six sequences responsible for platelet interaction, two in the E domain and two in each of the C-terminal parts of the fibrinogen molecule.

Adenosine Diphosphate↗