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M Furlan

Publications and source records attributed to M Furlan.

At least 127 records · Page 7Linked to original sources
PubMed1986

Von Willebrand factor-dependent agglutination of washed fixed human platelets by insoluble collagen isolated from bovine aorta.

Adult bovine aortic tissue was homogenized in a neutral phosphate buffer containing proteinase inhibitors. The insoluble residue was rehomogenized in Tris-buffered 6 mol/L guanidinium chloride (pH 7.4). An insoluble fibrillar protein, floating above the main pellet after recentrifugation, was harvested. This material agglutinated washed fixed human platelets in the presence of either normal human plasma or purified von Willebrand factor (vWF). No such reaction was seen when either buffer or plasma from patients with severe von Willebrand's disease was added instead. The extent of platelet agglutination was measured photometrically, similarly to the ristocetin cofactor assay. The agglutination reaction was strongest at neutral pH and was impaired after either addition of EDTA or previous digestion of the fibrillar material by collagenase or pepsin. By light microscopy platelets were seen to adhere onto isolated fibers. Amino acid composition, subunit polypeptides, substrate properties, and interaction with fibronectin of this fibrillar protein were comparable to those of collagen. Therefore, we tentatively denote the induction of platelet agglutination by vWF protein in the described test system as "vWF-collagen cofactor" activity. Comparison of this activity in 65 plasma samples, containing various concentrations of vWF, with ristocetin cofactor activity showed good correlation between results obtained in both tests (r = 0.91).

Agglutination Tests↗
PubMed1986

Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization.

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

Adult↗
PubMed1985

Reactivity of small molecular forms of human factor VIII/von Willebrand factor with botrocetin and anti-factor VIII-coated latex particles.

Two recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand's disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand's disease and its variants.

Antibodies↗
PubMed1985

Fibrinogen Bergamo I (A alpha 16Arg----Cys): susceptibility towards thrombin following aminoethylation, methylation or carboxamidomethylation of cysteine residues.

An abnormal fibrinogen, denoted as "fibrinogen Bergamo I", has been characterized. Its defect consists in an exchange of arginine by cysteine in position 16 of the A alpha-chain, thus corresponding to that found in a number of other fibrinogen variants. The abnormal fibrinopeptide A cannot be split off by thrombin from intact fibrinogen Bergamo I. We describe three different chemical modifications of the cysteine A alpha 16, i.e. aminoethylation, methylation and carboxamidomethylation, and their effects on the susceptibility of fibrinogen Bergamo I towards thrombin attack. S-aminoethylation of the A alpha 16Cys renders the peptide bond A alpha 16-17 cleavable by thrombin. Following methylation or carboxamidomethylation, the A alpha 19-arginyl bond becomes accessible for thrombin. The chemically modified extended fibrinopeptide A can be readily separated from the normal fibrinopeptide A by HPLC. The latter two modifications are suitable alternative procedures for detecting the molecular defect A alpha 16Arg----Cys of fibrinogen.

Amino Acids↗
PubMed1985

Inhibition of human T lymphocyte proliferation in vitro by commercial factor VIII concentrates.

Several investigators recently described a T lymphocyte subset abnormality (reversed helper/suppressor cell ratio) in haemophiliacs, especially following previous substitution therapy. We studied in vitro the potential effects of two commercial factor VIII concentrates on incorporation of 3H-thymidine by isolated human peripheral blood lymphocytes after stimulation with either phytohaemagglutinin or monoclonal antibody OKT3. Dose-related inhibition of thymidine incorporation was found for both concentrates but none using the purified main protein constituents, i.e. factor VIII, fibrinogen or fibronectin. Furthermore, the 'inhibitor' was completely removed by dialysis.

Acquired Immunodeficiency Syndrome↗
PubMed1984

[Desamino-8-D-arginine vasopressin nose drops in the treatment of mild hemophilia A and von Willebrand's disease].

To permit home treatment in our patients we investigated the response to intranasal administration of 260 micrograms DDAVP in 28 patients with either mild hemophilia A or von Willebrand's disease. 6 patients with mild hemophilia A responded, and 6 did not. In the group with von Willebrand's disease 6 responded well, 4 responded partially and 6 did not respond. Knowledge of the severity of the disease did not allow us to predict the response, and therefore every patient should be tested individually. Intranasal DDAVP in our patients was used with success for bleeding into the knee joint, muscle hematoma and hematuria.

Arginine Vasopressin↗
PubMed1984

Interactions of purified rat factor VIII/von Willebrand factor with rat and human platelets--effect of albumin and ristocetin.

Rat platelets do not respond to ristocetin in their own plasma nor do they aggregate in the presence of bovine or porcine factor VIII von Willebrand factor (F VIII R:WF) or human F VIII R:WF in presence of ristocetin. However, rat plasma supports ristocetin induced aggregation of washed human platelets. In this study we report on purification of rat F VIII R:WF from cryoprecipitate. Similarly to porcine or bovine material, purified rat F VIII R:WF induced aggregation of human washed fixed platelets. This effect was enhanced by addition of ristocetin and was not modified by addition of albumin. Rat washed platelets were aggregated by ristocetin in the presence of rat or human F VIII R:WF provided that high concentrations of ristocetin are added in a system essentially free of extraneous proteins. Increasing concentrations of albumin dramatically reduced the ability of ristocetin to aggregate rat platelets while human platelet aggregation by human or rat F VIII R:WF was only moderately affected. These studies show that rat F VIII R:WF can interact with rat and human platelets. The lack of response of rat platelets to ristocetin in their own plasma is most likely due to a low sensitivity of rat platelets to this drug and to an inhibitory activity of plasma proteins on this reaction.

Animals↗
PubMed1984

Isolation of small molecular forms of Factor VIII/von Willebrand factor from plasma.

Cryoprecipitated factor VIII/von Willebrand factor (FVIII/vWF), freed of fibrinogen by clotting with calcium and Defibrase, was chromatographed on Sepharose CL-2B. Fractions containing lower-molecular-weight forms of FVIII/vWF comprised coprecipitated plasma proteins of similar molecular weights. The major contaminants, fibronectin and IgM, were removed by affinity chromatography on gelatin- and anti-IgM-agarose, respectively. Finally, pure low-molecular-weight FVIII/vWF protein was harvested in the void volume fraction of a Sepharose CL-6B column. The smallest multimers had the size of the tetramer of the basic subunit chain of FVIII/vWF.

Blood Coagulation Factors↗
PubMed1983

[Fibrinogen Bern II: hereditary fibrinogen variant with amino acid substitution of arginine replaced by histidine in position 16 of the A alpha chain].

The number of described individuals with congenital dysfibrinogenemia continuously increases, but only a few fibrinogen variants have thus far been characterized with respect to their structural defect. Fibrinogen Bern II is a hereditary fibrinogen variant with impaired release of fibrinopeptide A (FPA) and a markedly prolonged coagulation time. It turned out that only half of the FPA was cleaved off at normal rate while the residual FPA was released much more slowly and incompletely, unless high thrombin concentrations were used. Amino acid analysis of normal and abnormal FPA revealed that the abnormal peptide, in contrast to the normal, contained histidine but hardly any arginine. It is therefore concluded that fibrinogen Bern II undergoes substitution of arginine in position 16 of the A alpha-chain by histidine. Thus, the structural alteration is identical with that of seven other recently described variants. The presence of 50% normal fibrinogen molecules provides the normal hemostasis in the heterozygous carriers of the Bern II-dysfibrinogenemia.

Afibrinogenemia↗
PubMed1983

[Hemostasis disorders with reduced activity of the von Willebrand factor in myeloproliferative syndromes].

Splenectomy performed in a patient with polycythaemia vera and incipient osteomyelofibrosis was complicated by severe postoperative bleeding. A transient decrease of factor VIII/von Willebrand factor (VIIIR:WF) was detected as a possible cause of the bleeding tendency. As a result of this observation the VIIIR:WF system was investigated in all patients with myeloproliferative disorders who are presently followed by this laboratory. Subnormal VIIIR:WF activity was found in 5 of 9 prospectively investigated patients with polycythaemia vera and in one of 12 patients with chronic myeloid leukemia. These results suggest that acquired defects of the VIIIR:WF system are, in addition to the well known functional abnormalities of blood platelets, quite common in myeloproliferative disorders, especially polycythaemia vera, and must be reckoned with before surgery in particular. When specific antibodies directed against VIIIR:WF have been ruled out, the etiology of the VIIIR:WF defect found in our patients remains unclear.

Blood Coagulation Factors↗
PubMed1983

Inhibition of fibrin polymerization by fragment d is affected by calcium, Gly-Pro-Arg and Gly-His-Arg.

Fibrinopeptides A and B were removed from purified human fibrinogen by bovine thrombin, whereas the snake venom protease batroxobin only split fibrinopeptide A from fibrinogen. Aggregation of the resulting desAB- and desA-fibrin monomers was evaluated by recording the turbidity of incubation mixtures. Fibrin assembly was strongly accelerated by increasing the calcium concentration from 10(-5) to 10(-3) M. Fragment D was obtained from fibrinogen by proteolytic degradation with plasmin in the presence of Ca2+. At a 4-fold molar concentration relative to fibrinogen, fragment D dramatically inhibited fibrin polymerization at up to 10(-4) M Ca2+. This anticlotting activity was, however, much less pronounced at 10(-3) M Ca2+. The thrombin clotting time, measured on human plasma, was prolonged by fragment D in a dose-dependent manner. In citrate-containing plasma, the fibrinogen clotting was significantly delayed by an equimolar concentration of fragment D. In barium sulfate-adsorbed oxalated plasma, containing 2.5 mM Ca2+, the same amount of fragment D hardly affected fibrin polymerization. We conclude that fragment D has no important anticlotting effect under physiological conditions. The synthetic peptide Gly-Pro-Arg, corresponding to the amino-terminal sequence of the fibrin alpha-chain, inhibited aggregation of both desA-fibrin and desAB-fibrin at 10(-3) M Ca2+. The inhibition of desAB-fibrin polymerization by Gly-Pro-Arg was abolished at 10(-5) M Ca2+. In addition, Gly-Pro-Arg depressed the anticlotting activity of fragment D at low calcium concentration. An analogue of the amino-terminus of fibrin beta-chain, Gly-His-Arg, strongly accelerated aggregation of desA-fibrin monomers, but only moderately enhanced polymerization of desAB-fibrin monomers at 10(-5) M Ca2+, both in the presence and in the absence of fragment D. This activating effect of Gly-His-Arg was abolished at 10(-3) M Ca2+. It is suggested that the binding of calcium, Gly-His-Arg, and possibly also Gly-Pro-Arg, induces a conformational change in fibrin monomers and thus accelerates the polymerization process.

Animals↗
PubMed1983

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels.

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.

Acrylamides↗
PubMed1982

Effect of calcium and synthetic peptides on fibrin polymerization.

Human fibrinogen was subjected to limited proteolytic attack by thrombin, batroxobin or Agkistrodon contortrix thrombin-like enzyme, yielding desAB-, desA- or desB-fibrin monomers, respectively. Turbidity curves demonstrated that, with all three enzymes, the polymerization process was strongly accelerated by increasing the calcium concentration from 10(-5) M to 10(-4) M. Synthetic peptide Gly-His-Arg (5 mM), an analogue of the aminoterminal sequence of fibrin beta-chain, inhibited aggregation of desB-fibrin monomers at physiological calcium concentration whereas it enhanced aggregation of desA- and desAB-fibrin monomers at calcium concentrations below 10(-4) M. On the other hand, Gly-Pro-Arg (1 mM) corresponding to the amino-terminus of fibrin alpha-chain, dramatically inhibited aggregation of both desA- and desB-fibrins, but it only moderately affected the polymerization of thrombin-induced monomers. We conclude that the observed effects of Gly-Pro-Arg and Gly-His-Arg are not due solely to their competition with fibrin amino-termini for the respective binding sites in the D-domain, but rather reflect conformational changes in fibrin monomers which affect the polymerization process.

Batroxobin↗