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Biomedical subjects

F Brosstad

Publications and source records attributed to F Brosstad.

At least 109 records · Page 6Linked to original sources
PubMed1986

Binding of various thrombin fractions to fibrin and the influence of AT-III on their adsorption.

Human thrombin with high affinity for fibrin was obtained by subjecting purified thrombin to affinity chromatography on Sepharose insolubilized fibrin monomers, after addition of a radioiodinated subsample of thrombin, molar ratio 1:600. As judged by radioprofiling of the electrophoretic distribution of high-affinity thrombin on 10 per cent polyacrylamide gel containing urea/SDS, the preparation consisted of 70 per cent alpha-thrombin, 28 per cent beta-thrombin and only 2 per cent gamma-thrombin. Although alpha-thrombin was bound more strongly to insolubilized fibrin monomers than the other subfractions, complete separation of the individual components could not be achieved. High-affinity thrombin was employed for studies on thrombin adsorption to polymerized fibrin, assuming equal behaviour of labelled and unlabelled thrombin. To avoid passive entrapment of thrombin within the fibrin meshwork at physiological pH, ionic strength and calcium concentration, the optimal fibrinogen concentration was found to be 2.94 umol/l. During such conditions, adsorption of thrombin to polymerized fibrin did not exceed 65 per cent of added thrombin, despite an increasing availability of fibrin-related thrombin binding domains obtained by reducing the thrombin concentration. Adsorption of thrombin to polymerized fibrin increased by 25 per cent when the ionic strength was reduced to 0.05 mol/l. These findings suggest the presence of thrombin subfractions with different affinities for polymerized fibrin. Aggregates of high-affinity thrombin formed during its preparation by affinity chromatography, but were prevented by adding polyethylene glycol (m.w. 6,000, final conc. 6.6 g/l). Such aggregates were not inactivated by AT-III, but could still adsorb to polymerized fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1986

Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immunoprecipitated glycoprotein IIb-IIIa complex.

To further investigate which parts of the fibrinogen molecule that are responsible for its binding to the fibrinogen receptor on human platelets, the following approaches were made: The glycoprotein IIb-IIIa complex (the putative fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100-extracts of platelets against antibodies to whole platelet proteins. Subsequently, the immunoplates were incubated with 125I-labelled, plasmin- or CNBr-cleaved fibrinogen fragments (pre-X,X,Y,D,Degta,Efg,N-DSK) or fibrin fragments (E1,N-dsk), characterized by partial sequenation. The immunoplates were exposed to X-ray films, and binding of the fragments to the glycoprotein IIb-IIIa complex was examined. The findings were compared to the results obtained from studies on binding of the same fragments to intact gel-filtered platelets after ADP-stimulation. The following conclusions were made: All fragments except Efg and Degta bound to the immunoprecipitated GPIIb-IIIa complex as well as to ADP-stimulated platelets suggesting that at least two sequences in the E domain and one in each of the D domains of fibrinogen are involved in binding to the platelet receptor. The GPIIb-IIIa complex is the only surface-located platelet antigen that binds fibrinogen and the aforementioned fragments. The binding of the fragments to the receptor is dependent on divalent cations.

Antibodies↗
PubMed1986

Binding properties on Sepharose insolubilized fibrinogen and fibrin, of various species of fibrinogen and fibrin solubilized in plasma.

Radiolabelled tracers of fibrinogen, fibrin des-AA and fibrin des-AABB were solubilized in recalcified, prothrombin depleted plasma, adding either 125I-fibrin des-AA or 125I-fibrin des-AABB together with 131I-fibrinogen, and subsequently subjected to affinity chromatography, utilizing short columns of Sepharose insolubilized preparations of fibrinogen, fibrin des-AA and fibrin des-AABB, respectively. Two naturally occurring fibrinogen species, of high molecular weight (HMW; m.w. 340.000) and of low molecular weight (LMW; m.w. 305.000) exhibited similar binding characteristics, as judged by adsorption and desorption experiments. In subsequent studies all tracer preparations were derived from HMW-fibrinogen. Sepharose insolubilized fibrinogen favoured the adsorption of soluble fibrins as compared to fibrinogen in solution; the adsorption of soluble des-AA fibrin was similar to that of soluble des-AABB fibrin. To insolubilized fibrin, adsorption of soluble tracers of fibrinogen and fibrins increased considerably, and soluble fibrins were no longer preferentially adsorbed. The latter observation was supported by similar desorption characteristics of these tracers. These findings may indicate that the E-domains of soluble fibrin become largely inaccessible to the D-domains of Sepharose insolubilized fibrinogen, probably due to complexing fibrinogen in plasma. Furthermore, adsorption was largely related to the a-epitope of insolubilized fibrin.

Animals↗
PubMed1986

Visualization of von Willebrand factor multimers by enzyme-conjugated secondary antibodies.

A method for visualization of the multimeric forms of von Willebrand Factor (vWF) in plasma and platelets is described. The method is based upon: 1) Separation of the vWF multimers by SDS-agarose electrophoresis, 2) Subsequent blotting of the vWF multimers onto nitrocellulose, 3) Immunolocalization and visualization of the vWF pattern by the sequential incubation of the blot with primary vWF antiserum, peroxidase- or beta-galactosidase-conjugated secondary antibodies and a relevant chromogenic substrate.

Antibodies↗
PubMed1986

Increased binding to ADP-stimulated platelets and aggregation effect of the dysfibrinogen Oslo I as compared with normal fibrinogen.

Interactions of the dysfibrinogen Oslo I with platelets were investigated. This fibrinogen is a B beta-chain variant with faster than normal fibrin monomer polymerization. Fibrinogen Oslo I acted more efficiently in ADP-induced platelet aggregation, and bound to gel-filtered platelets with a higher affinity constant than did normal fibrinogen. At all concentrations more fibrinogen molecules became bound per platelet with the dysfibrinogen than with normal fibrinogen, both when the fibrinogens were tested separately or as a mixture using 125I or 131I to label the two types. At high concentrations this was probably due to ligand polymerization of the dysfibrinogen. These observations indicate that the increased cofactor function in platelet aggregation may be related to the increased affinity of the dysfibrinogen for the platelets.

Adenosine Diphosphate↗
PubMed1985

Polymerization properties of two normally circulating fibrinogens, HMW and LMW. Evidence that the COOH-terminal end of the a-chain is of importance for fibrin polymerization.

The plasma fibrinogen fractions HMW (mw 340,000) and LMW (mw 305,000) were prepared from purified (beta-alanine precipitated) fibrinogen by step-wise precipitation with ammonium sulfate. The thrombin clotting times were 14" and 20" respectively. The enzymatic phase of coagulation, measured as release of fibrinopeptide-A during incubation with thrombin, was found to be identical for HMW and LMW. Polymerization was studied by light scattering (at 605 nm) using preformed monomers (des-AA and des-AABB) prepared from HMW and LMW in the presence of 3.3 M urea by incubation with thrombin (100 NIH U/ml final conc.) and reptilase (1 U/ml final conc.). The HMW-monomers polymerised at a substantially higher rate than the corresponding LMW-monomers. Thus, the prolonged clotting time of LMW was explained by retarded polymerization. It is suggested that the -COOH terminal end of the a-chain, containing the molecular difference between HMW and LMW, is of importance for polymerization. Furthermore, the release of fibrinopeptide B (des-AABB-monomers) improved polymerization properties in HMW as well as in LMW, and all types of monomers polymerised more rapidly in the presence of Ca++.

Fibrin↗
PubMed1985

Clearance characteristics of des-AA fibrin and des-AABB fibrin, and thrombus-related uptake of des-AABB fibrin as compared to fibrinogen.

The following paper presents a short review of previous studies relating to the behaviour in man of radiolabelled fibrins des-AA and des-AABB, as compared to that of radiolabelled fibrinogen. Des-AA fibrin was eliminated with a half-life of 30 to 60 min in eight healthy controls, but its half-life was substantially shorter in eight fibrinaemic patients with no demonstrable fibrinolysis. Clearance of des-AABB fibrin was studied in thirteen patients with established venous thrombosis, all subjected to a concomitant fibrin(ogen) uptake test. There was no essential difference in its half-life in patients with a positive fibrinogen uptake test (n = 7) as compared to those with a negative test (n = 6). The metabolic half-life of des-AABB fibrin was 10 +/- 3.5 hrs. The uptake of labelled des-AABB fibrin by thrombi was similar to that of labelled fibrinogen during the first hours after injection, but only fibrinogen could reflect a continuous build-up of thrombi, due to its longer survival time.

Fibrin↗
PubMed1985

Common structural genes for platelet and plasma fibrinogen.

Fibrinogen from plasma was compared with fibrinogen from platelets using two-dimensional electrophoresis. The source of platelet fibrinogen was isolated alpha-granules, thrombin- and collagen-released platelet material. The B beta- and gamma-chains from the different sources showed similar two-dimensional patterns, while gamma'-chains were not observed in platelet fibrinogen preparations. Furthermore, the A alpha-chain could hardly be identified in platelet preparations. When individual fibrinogen was studied in persons heterozygous for genetic B beta- and gamma-chain variants, the two-dimensional variant pattern could be demonstrated in plasma fibrinogen as well as in platelet fibrinogen. This observation strongly indicates that the structural genes for plasma and platelet fibrinogen B beta- and gamma-chains are identical.

Blood Platelets↗
PubMed1984

Quantitation of factor XIII by SDS polyacrylamide gel electrophoresis.

An electrophoretic method for the determination of f.XIII, based on the capability of f.XIIIa to cross-link fibrin clots (5) was studied, and the great sensitivity of the method confirmed. Thus, with suitable technical conditions, f.XIII activities less than 0.1% of that in normal human plasma could be detected. The results obtained in plasma from various patients and in f.XIII concentrates (cryoprecipitates) corresponded well with those obtained with the dansylcadaverine method (3) and with the urea solubility test, with two exceptions: In a patient with severe congenital f.XIII deficiency, only the present method was sensitive enough to detect any f.XIII-activity (about 0.1%), and in a patient with an inhibitor against f.XIIIa, the dansylcadaverine method failed to detect this. The present method is too laborious for routine screening, but is recommended as an alternative references method. It may prove especially suitable to detect minute amounts of f.XIII.

Cadaverine↗
PubMed1984

Thrombus-related uptake and vascular clearance of 131 I-fibrin des-AABB as compared to 125 I-fibrinogen in patients with established venous thrombosis.

Thrombus-related uptake of 131 I-fibrin des-AABB has been compared to that of 125 I-fibrinogen in 13 patients with established venous thrombosis. Both tracers originated from a common pool of beta-alanine precipitated fibrinogen. Scan-recordings were performed as a radiofibrin (ogen) uptake test. Uptake characteristics of des-AABB fibrin were similar to those of fibrinogen, when measured as percentage of concomitant radioactivity over the heart. Due to its longer circulation time, fibrinogen was superior to fibrin des-AABB for the detection of venous thrombi. Circulating des-AABB fibrin was cleared biphasically, with an initial rapid decline followed by a gradual exponential decrease. Mean half-lives were 5.5 +/- SD 3.5 hr and 10 +/- SD 3.5 hr, respectively. The elimination rates were uninfluenced by thrombus activity, as judged by the fibrin(ogen) uptake test. Metabolic half-life of fibrinogen in the total material was 62 +/- SD 19 hr. Dissociation of fibrinogen and soluble des-AABB fibrin clearance rates was evident, describing their own, independent elimination patterns, probably reflecting different clearing mechanisms.

Fibrin↗
PubMed1983

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography.

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.

Animals↗
PubMed1983

Complex-formation between the fibrin-derived plasmic fragments DD and E demonstrated by crossed immunoelectrophoresis.

The formation of a complex between the fibrin fragments DD and E was studied by crossed immunoelectrophoresis using antibodies against human fibrinogen. The complex formation was seen by a common electrophoretic migration of the DD-fragment and part of the E-fragments. This effect was abolished by a further incubation with plasmin of the preparation containing the (DD) E-complex. This also led to an anodal shift in migration of the E-fragment indicating a transfer from E1 to E3.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

A new dysfibrinogenemia: fibrinogen Oslo IV.

A family with dysfibrinogenemia is described. The abnormal fibrinogen occurred in three successive generations indicating a dominant hereditary pattern. Thrombin and reptilase times were about twice the normal value. This was shown to be caused by a polymerization defect, fibrinopeptide release being normal. Platelet aggregation was undisturbed, indicating normal platelet-fibrinogen binding. The bleeding time was normal and there was no bleeding tendency. However, an obscure recurrent pulmonary ailment may, or may not, be related to the dysfibrinogenemia. The abnormal fibrinogen was tentatively termed Oslo IV.

Amino Acid Sequence↗
PubMed1983

Demonstration of a new glycoprotein Ib-related component in platelet extracts prepared in the presence of leupeptin.

The water-soluble protein glycocalicin is generated during platelet lysis by a proteolytic attack on the integral membrane glycoprotein GP Ib. However, only small amounts of glycocalicin are formed when platelets are solubilized by 1% Triton X-100. Crossed immunoelectrophoresis of such extracts using an antiserum to glycocalicin, shows a continuous immunoprecipitate consisting of two peaks, one representing glycocalicin and the other GP Ib. When leupeptin was present during solubilization, subsequent immunoelectrophoresis revealed yet another GP Ib-related component represented by a third, slow-migrating peak of the immunoprecipitate. During incubation of platelets with dibucaine followed by solubilization in the presence of leupeptin, a gradual transformation of this new form of GP Ib into the previously defined one took place prior to the formation of glycocalicin. An increase followed by a decrease in the agglutination response of the platelets to bovine von Willebrand factor occurred concomitant with these transformations. SDS-polyacrylamide gel electrophoresis of Triton X-100 extracts of platelets did not reveal any difference in the size of GP Ib whether or not leupeptin had been present during the solubilization.

Animals↗
PubMed1983

Platelet factor XIII is an active enzyme after solubilization and crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of platelets against antiplatelet antibodies has proved to be a valuable tool in the study of platelet proteins (1-8). The advantage of this separation system is that the proteins are separated under nondenaturating conditions and thus to some extent would be expected to maintain their functional properties. Previously, the binding of several proteins to immobilized thrombin (5) and immobilized heparin (9) during crossed immunoelectrophoresis of platelet proteins solubilized in a Triton X-loo-containing buffer has been described. Furthermore, it has been demonstrated that fibrinogen is able to bind to immunoprecipitates containing the glycoprotein IIb-IIIa-complex (7). These studies indicate that the proteins contained in the immunoprecipitates represent biologically active entities. In the present study we provide direct evidence for this by demonstrating enzymatic activity associated with the immunoprecipitate containing Factor XIII in immunoplates obtained after crossed immunoelectrophoresis of solubilized platelets against anti-platelet antibodies.

Blood Platelets↗
PubMed1983

Molar antithrombin concentration in normal human plasma.

Crude, commercial thrombin preparations and purified bovine thrombin were incubated with normal human reference plasma and the amount of thrombin inactivated was calculated. 1 ml of human plasma inactivated 140-193 NIH U of the various crude thrombin preparations. In the presence of heparin, a lower thrombin-inactivating capacity of plasma was confirmed using crude thrombin, but this phenomenon was less pronounced with the purified thrombin preparation. The molar concentration of the purified bovine thrombin was determined by active site titration. Comparing with protein concentration (A280), this preparation was 92% pure. 1 ml of human plasma inactivated 2.57 mumol of thrombin in the absence of heparin, and 2.50 mumol with heparin. Assuming 1:1 stoichiometry in the thrombin-antithrombin reaction, these results suggest that the concentration of antithrombin in the pooled reference plasma is approximately 2.57 mumol/l or 0.15 g/l.

Animals↗