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M Furlan

Publications and source records attributed to M Furlan.

At least 145 records · Page 8Linked to original sources
PubMed1981

[Fibrinogen Bern I and fibrinogen Bern II: 2 hereditary fibrinogen variants with diverse biochemical properties].

Two inherited fibrinogen variants, fibrinogen Bern I and fibrinogen Bern II, have been partially characterized. The markedly prolonged thrombin clotting times observed with both variants result from impaired fibrin monomer aggregation. Complete correction of this polymerization defect at physiological calcium concentrations has been achieved for fibrinogen Bern I. This variant also exhibits a charge abnormality in the gamma-polypeptide chain, an increased sialic acid content and defective calcium binding by the C-terminal part of the molecule. Fibrinogen Bern II, on the other hand, exhibits a defect which, although located at a site important for polymerization, is not influenced by the binding of calcium ions.

Binding Sites↗
PubMed1981

Von Willebrand activity of low molecular weight human Factor VIII increases by binding to gold granules.

Human factor VIII/von Willebrand protein is a population of multimers which vary in size but contain apparently identical subunits. Large-molecular-weight forms possess higher ristocetin cofactor/von Willebrand activity than the native smaller oligomers. Disulfide reduction of large factor VIII multimers results in progressively decreasing molecular size and a loss of ristocetin cofactor activity. Small molecular forms of factor VIII were adsorbed onto gold granules (average diameter 20-30 nm) and thereby increased their ristocetin cofactor activity. The amount of adsorbed material and the extent of activation were dependent on the pH of the coiled suspension. The maximum recovery of von Willebrand activity was observed at pH 4.75. Aggregation of fixed human platelets by factor VIII-coated gold particles was dependent on ristocetin concentration and was not competitively inhibited by unbound low-molecular-weight factor VIII. These results suggest that the subunits of the native small factor VIII species possess potential binding affinity for platelet receptors, which is manifested following formation of large factor VIII polymers. We conclude that an optimal size of remarkably high molecular weight is required for efficient aggregation of platelets by factor VIII as occurs during the primary phase of hemostasis.

Adsorption↗
PubMed1981

Fractionation of individual, biologically active factor VIII multimers.

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.

Antigens↗
PubMed1981

Binding of bovine factor VIII-coated colloidal gold particles to receptors on platelet membranes.

Bovine factor VIII/platelet aggregating factor was adsorbed into gold granules and the protein-gold complex added to either formalin-fixed or fresh washed human platelets. Following aggregation, binding of gold granules to the platelets was measured by monitoring the optical density of colloidal gold remaining in the supernatant. Scatchard analysis of binding data indicated that multiple classes of binding sites were present. The number of high affinity binding sites per formalin-fixed platelet depended on the concentration of ristocetin: 420 gold granules were calculated to bind at 1.4 mg/ml of ristocetin, 610 at 0.6 mg/ml of ristocetin and 875 when no ristocetin was added. Fresh washed platelets bound 1350 granules per cell in the absence of ristocetin. We conclude that during platelet aggregation, induced by bovine factor VIII, the binding sites on the platelet surface are only partially occupied.

Animals↗
PubMed1981

Binding of colloidal gold granules, coated with bovine factor VIII, to human platelet membranes.

Platelet aggregating factor (PAF) activity is preferentially associated with the larger molecular forms of bovine factor VIII and diminishes considerably after dissociation into smaller oligomers by mild disulphide reduction. PAF activity was regained by the binding of small factor VIII oligomers to colloidal gold granules with a mean diameter of 23 nm. In contrast, adsorption of large factor VIII polymers onto gold particles resulted in partial loss of PAF activity. Thus, an optimum multimeric size of bovine factor VIII appears to be required for the maximal expression of PAF activity. Gold granules, coated with reduced factor VIII, can be used as an electron-dense label. Their interaction with surface receptors for bovine factor VIII on either viable or formalin-fixed human platelets was demonstrated by transmission and scanning electron microscopy.

Animals↗
PubMed1981

Factor VIII activity in chronic renal disease.

F VIII coagulant, F VIII-related antigen and F VIII ristocetin cofactor activity were significantly increased in 68 patients with various chronic renal diseases. All three F VIII functions correlated generally well with each other. A striking relationship between some F VIII activities and serum creatinine was detectable in patients with glomerulonephritis and kidney transplants, with mild or moderate renal insufficiency. This correlation was no longer present in terminal renal failure. The results suggest that in initial stages of renal disease elevated F VIII levels may be attributable to glomerular endothelial damage. In terminal renal failure, however, increased F VIII concentrations seem to result from nonspecific causes related to uremia such as acute phase reactions.

Adolescent↗
PubMed1980

[Increased von Willebrand activity of low molecular-weight factor VIII following binding to gold granules].

High molecular weight factor VIII was partially reduced with beta-mercaptoethanol. Disulfide reduction resulted in progressive dissociation of the multimeric protein with a concomitant decrease in von Willebrand activity. The binding of reduced small factor VIII oligomers to gold particles with an average diameter of 20--25 nm led to marked "activation" of their von Willebrand activity. A similar increase in activity was also observed following adsorption of native small molecular weight forms of factor VIII to gold granules. Binding of initially highly active high molecular weight factor VIII to gold granules resulted in inhibition of von Willebrand activity, probably by formation of superaggregates. The platelet aggregating function of factor VIII in the circulation appears to depend largely on the size of this multimeric protein, which must be in the range of several millions for its maximum expression.

Adsorption↗
PubMed1980

Studies on factor VIII-related protein. IV. Interaction of galactose-specific lectins with human factor VIII/Von Willebrand factor.

Factor VIII of human cryoprecipitate was purified and fractionated on Sepharose CL-2B into three fractions of progressively decreasing multimer size and ristocetin cofactor activity. Following complete disulfide reduction, the resulting subunits were electrophoresed on 3% polyacrylamide gels and subsequently stained with two galactose-specific, fluorescein-labelled lectins from Ricinus communis (RCAI and RCAII). Measurements of fluorescence indicated that the reduced chains, derived from the largest factor VIII multimers, have stronger binding affinity for both lectins than those obtained after reduction of smaller factor VIII species. Ristocetin cofactor activity of purified factor VIII was competitively inhibited by both Ricinus lectins and by concanavalin A. RCAI-lectin was found to be a considerably more efficient inhibitor than RCAII or concanavalin A. Following removal of sialic acid from factor VIII, the inhibiting effect of RCAII-lectin was markedly potentiated, probably by exposing additional galactose residues, some of which must be located close to the ristocetin cofactor 'active site' of factor VIII. Ristocetin cofactor activity was also strongly inhibited with RCAII-lectin for binding sites which are located on the surface factor VIII multimers. Our results suggest that RCAI-lectin, which contains galactose-specific binding sites per molecule, and anti-factor VIII antibodies inhibit ristocetin cofactor activity by crosslinking and aggregation of factor VIII multimers.

Antibodies↗
PubMed1979

[Is there a correlation between sugar content, thrombocyte aggregating activity and molecular size of factor VIII?].

Human and bovine factor VIII, isolated from cryoprecipitates of fresh plasma by gel filtration on Sepharose CL-2B, gave similar elution patterns and showed comparable distribution of oligomers on SDS agarose electrophoretic gels. The carbohydrate content of individual factor VIII bands, measured by reaction with dansyl hydrazine or binding of glucose/mannose specific concanavalin A, was not directly related to the size or von Willebrand activity of factor VIII oligomers. Staining of disulfide-reduced factor VIII subunits, in polyacrylamide gels, with galactose-specific fluorescein-labelled Ricinus communis lectins, showed an increased binding affinity with increasing size and von Willebrand activity of the parent factor VIII. The von Willebrand activity was strongly inhibited by reaction with Ricinus RCAI lectin, whereas concanavalin A inhibited platelet aggregation only at concentrations above 1 mg/ml. These results suggest that galactose residues are involved in the aggregation of platelets by factor VIII.

Animals↗
PubMed1979

Studies on factor VIII-related protein. III. Size distribution and carbohydrate content of human and bovine factor VIII.

Human and bovine factor VIII were isolated from cryoprecipitate of fresh frozen plasma by gel filtration on Sepharose CL-2B. The elution diagrams and SDS-agarose electrophoretic analysis of eluted fractions show no significant differences in the size-distribution of factor VIII aggregates between the two species. Agarose gels were stained for carbohydrate by two methods: (1) the dansyl hydrazine reaction following oxidation with periodic acid and (2) staining with fluorescein-labeled concanavalin A. Results of both procedures indicate that in human factor VIII neither the size distribution nor its ristocetin cofactor activity are related to carbohydrate content. Bovine factor VIII contains slightly less sugar than the human preparation as judged from the relative dansyl hydrazine staining intensities. In contrast to human factor VIII, the binding affinity for concanavalin A of bovine factor VIII was gradually decreased with increasing aggregate size. This finding suggests an impaired accessibility of reactive sugar residues in large aggregates of bovine factor VIII.

Animals↗
PubMed1979

A thiocholine ester of cinnamic acid inhibits crosslinking of fibrin without specific binding to donor lysines.

In the presence of activated factor XIII, 2-diethylbenzyl-aminoethylthiol-14C-transcinnamate bromide completely inhibited the crosslinking of fibrin. However, all three fibrin chains bound the cinnamic acid, and, in the alpha- and gamma-chains, the binding of label was not restricted to the crosslinking donor sites as might be expected. Furthermore, even in the absence of activated factor XIII, fibrinogen and fibrin incorporated cinnamic acid. Thus, as well as reacting with the functional thiol group of factor XIII, the thiocholine ester of cinnamic acid is incorporated non-specifically throughout the fibrinogen and fibrin subunit chains. The thiocholine ester used differs in this respect from dansyl cadaverine which is incorporated enzymatically and exclusively to the (acceptor) sites involved in crosslinking. Thiocholine ester of cinnamic acid cannot be used as a label for localization of specific crosslinking donor sites.

Chemical Phenomena↗
PubMed1979

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.

Disulfides↗
PubMed1979

Studies on factor VIII-related protein. II. Estimation of molecular size differences between factor VIII oligomers.

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.

Chromatography, Gel↗
PubMed1979

Preparation of factor VIII-deficient plasma by immunoadsorption.

An immunoadsorbent was prepared by coupling rabbit and human antibodies against human factor VIII to Sepharose CL-2B. The resulting insoluble antibodies completely removed factor VIII and fibrinogen from normal human citrate plasma. Other coagulation factors were satisfactorily recovered in the eluted plasma. Following addition of fibrinogen, the factor VIII-deficient plasma was used for calibration of the one-stage factor VIII assay and compared with the hemophilic plasma. Parallel straight lines were obtained in the log-log plot against VIII:C, indicating that the artificial reagent can be used as a substitute for hemophilic plasma in determination of factor VIII procoagulant activity. The immunoadsorbent can be regenerated and repeatedly used for affinity chromatography binding of factor VIII.

Animals↗
PubMed1977

Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).

The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.

Blood Coagulation↗