Bernt Ly

Stabilization of soluble fibrin/fibrinogen complexes by Fibrin Stabilizing Factor (FSF)

Abstract When fibrinogen was incubated with traces of thrombin, in the presence of FSF and calcium, and thrombin subsequently inhibited with Hirudin to prevent clotting, soluble fibrinogen derived oligomers, resistant to SDS-urea, were formed. The oligomers, separated from unpolymerized fibrinogen by agarose gel chromatography, demonstrated no species migrating as native fibrinogen, when subjected to SDS-gel electrophoresis in the presence of urea. Electrophoresis after reduction with mercaptoethanol demonstrated the oligomers to be y-y crosslinked, whereas no alfa chain crosslinking appeared. Thus, prior to visible gelation, oligomers, covalently linked by FSF, through y-chains alone, were formed. Thrombin activated FSF (thrombin subsequently inhibited with Hirudin) also oligomerized fibrinogen directly. Subsequent gel chromatography separated a fibrinogen dimer. When electrophoresed after reduction this contained four subunits, the alfa A, the beta B and approximately equal amounts of y- and y-y dimer, suggesting that one of the two y-chains of each fibrinogen molecule was intercrosslinked.

Incorporation of fibrinogen into soluble fibrin complexes

Abstract Fibrinogen was incubated with traces of thrombin, and the reaction stopped with hirudin prior to visible gelation. When I125-labelled fibrinogen was added to the mixture before chromatography on Sepharose 4B, labelled fibrinogen eluted not only in the main fibrinogen peak, but also in the earlier fractions containing high molecular weight complexes. This implies that fibrinogen not acted upon by thrombin, may form complexes with soluble fibrin. The complexes detected by gel chromatography could not be demonstrated by SDS-urea polyacrylamide gel electrophoresis, indicating that non-covalent bonds are involved.

Purification of human factor IX.

Abstract Factor IX was isolated from human plasma by a six-step procedure employing adsorption to DE-52 cellulose in batches, aluminium hydroxide adsorption, affinity chromatography on heparin-coupled Sepharose, preparative polyacrylamide disc gel electrophoresis, immunosorbent absorption, and DE-52 cellulose chromatography. Determination of factor IX activity and antigen revealed a specific activity of about 290 U per absorbancy unit at 280 nm. Analytical polyacrylamide disc gel electrophoresis showed one major and three to four minor bands with lower electrophoretic mobility. Disc gel electrophoresis in the presence of 10 M urea gave a double major band and two additional minor bands with higher electrophoretic mobility. One major band was observed in SDS polyacrylamide gel electrophoresis, and no change in mobility took place after reduction.

Molecular aspects of the clottable proteins of human plasma during fibrinogenolysis.

Abstract During incubation of human plasma with urokinase the clottable proteins were harvested by clotting with thrombin in the presence or absence of EDTA. SDS-gel electrophoresis after reduction of the ‘fibrins’ demonstrated a gradual loss of intact α chains concomitant with prolongation of the thrombin clotting time. Thus, the clottable proteins had become virtually devoided of intact A α chains when the thrombin clotting time was doubled (20–40 seconds), whereas the amount of clottable proteins was lowered by 20 per cent only. Only limited amounts of FDP could be detected in serum at this stage. Thus, the prolonged thrombin clotting time during the initial proteolysis reflects delayed polymerization of clottable derivatives with partially degraded A α chains, rather than interference of unclottable FDP with the clotting of native fibrinogen. N-terminal analysis of the ‘fibrins’ from UK-incubated plasma demonstrated the appearance of N-terminal alanine and a reduction in the molar content of N-terminal glycine. This finding most likely reflects release of a fragment containing fibrinopeptide B from the N-terminal part of the B β chains. Hence, fibrinogenolysis in plasma initially seems to follow the degradation sequence observed with purified fibrinogen.

Some aspects of the bonds interlinking soluble fibrin complexes in human plasma

Abstract I125 labelled fibrinogen was used to study the appearance of soluble fibrin complexes in thrombin incubated citrated plasma or in glass activated native plasma. Polyacrylamide gel (4%) electrophoresis demonstrated that approximately 7% of the plasma fibrinogen had been converted to slower moving species, eg. soluble fibrin complexes. Only a minor part of these complexes, with mobility as a fibrinogen derived dimer, persisted upon electrophoresis in the presence of 5 M urea. Also, agarose gel chromatography (Seph. 4 B) revealed the formation of high molecular weight fibrin/fibrinogen derivatives, which disappeared when chromatography was performed in the presence of 5 M urea. Thus, the major part of the soluble fibrin complexes in plasma was not resistant to urea, indicating that mainly non covalent bonds were involved. This implicates only a minor part of such complexes in a plasma milieu to be cross-linked through the action of the Fibrin Stabilizing Factor.

Radioiodinated human fibrinogen. In vitro effects of increasing iodination

Abstract Fibrinogen preparations with increasing molar contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICI method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule. Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine. Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isoelectric focusing. Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

Immunological typing of acute leukemias: Immunoenzymatic staining of fixed cells compared with immunofluorescence staining of unfixed cells in suspension

A panel of 14 monoclonal antibodies (McAb) against hematopoietic cell surface antigens was applied on mononuclear blood or bone marrow cells from 40 cases of acute leukemia in order to compare immunoenzymatic staining (IE) (alkaline phosphatase) of fixed cells with immunofluorescence staining (IF) of unfixed suspended cells. According to the immunological results, 25 cases were phenotyped as ALL and 15 cases as AML. Cases with blast crisis secondary to chronic myelogeneous leukemia (CML‐BC) were not represented in this series. In all ALL cases the two methods gave an identical antigenic distribution. In 20 our of 21 cases of non‐T‐cell ALL, a B‐cell progenitor origin was demonstrated by a positive staining reaction with the anti‐CD 19 McAb AB1 or HD37, and in 10 cases additionally with the anti‐CD20 McAb B1 or 1F5. In contrast to the results obtained with IF, IE revealed a poor preservation of the AB1 epitope on CD19, whereas the HD37 epitope was equally well demonstrated by both methods. In 15 cases of AML the distribution of positive versus negative cells with IE or IF was identical for all McAb except J5 (anti‐CALLA) (CD10) and Bl (CD20). Thus, 10/15 AML cases expressed CALLA with IE compared to 2/15 with IF. The corresponding figures for Bl were 5/15 and 0/15, respectively. Accordingly, normal myeloid precursor cells were CALLA‐positive with IE but negative with IF. The discrepancy probably reflects the fact that, whereas both intracytoplasmatic and membrane‐bound antigens are exposed in IE, only the latter are in IF. If the alteration of antigenic accessibility after fixation is considered, IE can safely be used for immunophenotyping of acute leukemia.

Characterization of soluble, FSF-stabilized fibrin complexes with special reference to the role of fibrinogen

Abstract Fibrinogen was incubated with traces of thrombin in the presence of calcium, and thrombin subsequently inhibited with Hirudin. When activated FSF was added, soluble fibrinogen derived oligomers, resistant to SDS-urea were formed. The relative content of fibrinogen in the oligomers was studied by adding I-125 labelled fibrinogen prior to the addition of FSF and recording the incorporation of radioactivity into the stabilized oligomers, separated by SDS-gel electrophoresis. Since 25 % of the protein was converted to stabilized oligomers and only 3 % of the labelled fibrinogen incorporated, the oligomers predominantly consisted of fibrin. N-terminal analysis of these oligomers demonstrated a molar content of N-terminal glycine of 1.4 moles. Assuming 2 moles of N-terminal glycine to be generated per mole of fibrinogen when both fibrinopeptides A are removed, this would give allowance for less than 30 % fibrinogen molecules in the oligomers.

Segregation of Malignant Hematological Disease in Families with Malignant Lymphoma

Familial malignant lymphoma, viz. more than two cases of malignant hematological disease in a family of which one diagnosis is malignant lymphoma, was seen in 43 (37 per cent) of the families in our database of familial malignant hematological disease. Genealogical examination of the 43 pedigrees after multiple ascertainment showed an equal amount of vertical transmissions (affected parent-offspring and grandparent-parent-offspring combinations) and non-vertical transmissions (affected uncle, aunt-nephew, niece, cousin combinations) without evident Mendelian pattern and no significant difference between observed and expected patrilineal and matrilineal lines in spite of a marked predominance of males. A marked pleiotropic diversity of involved diagnoses comprised 57 (93 per cent) lymphoproliferative- and 4 (7%) myeloproliferative diseases. Both Hodgkin’s lymphoma, non-Hodgkin’s lymphoma apart from the diffuse large B-cell lymphoma and chronic lymphocytic leukemia had a strong mutual association, and a weaker yet significant association to multiple myeloma and to diffuse large B-cell lymphoma. Compared with the number of patients in the population extracted from the crude age-adjusted incidences, the observed number of patients in familial disease was significantly higher interpreted as a stronger expression of congenic susceptibility among family members with reservations related to different environmental factors. Signs of anticipation in all combinations but no birth order effect were observed. It is discussed that an epigenetic parental genomic imprinting as a modifier for the segregation of linked susceptibility loci theoretically would bring about a similar pattern with male predominance and pleiotrophic diversity of diagnoses away from any Mendelian expectation.

Familiær forekomst av kronisk lymfatisk leukemi i Norge

BACKGROUND The only known risk factor for chronic lymphocytic leukaemia (CLL) is occurrence of the disease in close relatives. The aim of this study was to determine the frequency of familial chronic lymphocytic leukaemia. MATERIAL AND METHOD All patients with chronic lymphocytic leukaemia notified to the Cancer Registry in the period 1.10.2007-31.12.2009 were asked to report occurrences of malignant disease in siblings, parents, grandparents and children. The information about malignant haematological disease was verified with the Cancer Registry. RESULTS We found malignant haematological disease in close relatives of 42 of the 236 included patients (18%). CLL and lymphoma were the most common diagnoses. On average, 16 family members were identified in each family. The relative risk of developing CLL was six times higher in those who had close relatives with the disease (16 of a total of 3,776 family members) than among those who did not have close relatives who were affected (76 cases among 107,223 family members of 38,159 control subjects). The increased risk of disease was also associated with other lymphoproliferative diseases. With patrilinear, but not matrilinear inheritance, we found a birth order effect, with affection of younger men in a group of siblings, while the eldest escaped. INTERPRETATION Malignant haematological disease is common in the family members of patients with CLL. CLL is the most common disease, but there is extensive pleiotropy.