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Biomedical subjects

M Furlan

Publications and source records attributed to M Furlan.

At least 163 records · Page 9Linked to original sources
PubMed1976

Bleeding disorder with abnormal wound healing, acid-soluble clots and normal factor XIII.

An unusual bleeding disorder clinically resembling factor XIII deficiency is presented. The only detectable coagulation abnormality was rapid clot dissolution in 1% monochloroacetic acid. This abnormality was ascribed to the sustained increase of a pepsin-like plasma protease which is activated at low pH. Asystematic search for similar phenomena revealed that massive blood transfusion may also enhance plasma-clot solubility in acid, possibly by release of a red cell protease. We conclude that the acid clot solubility test is not a specific indicator of factor XIII deficiency, but this simple assay is recommended for further studies of acid plasma protease activity. The diagnostic relevance and pathophysiologic importance of increased pepsin-like activity in plasma remain to be elucidated.

Acetates↗
PubMed1976

Clottability and cross-linking reactivity of fibrin(ogen) following differential release of fibrinopeptides A and B.

Human fibrinogen was treated at pH 6.0, 7.3 and 9.0 with thrombin, batroxobin (thrombin-like fraction of Bothrops atrox venom) or an extract of the venom from Ancistrodon contortrix contortrix. These three enzymes released the NH2-terminal fibrinopeptides A and B at different rates. Thrombin-free, preactivated factor XIII (factor XIIIa) was added to incubation mixtures to stabilize resulting fibrin(ogen) aggregates. Cross-linking of gamma-chains and the size of covalently linked fibrin-fibrinogen oligomers were studied in an early stage of fibrinopeptide cleavage using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Batroxobin (pH 7.3) and thrombin (pH 6.0) preferentially released fibrinopeptide A, and resulting fibrin aggregates became rapidly insoluble. However, when fibrinopeptide B was removed with the contortrix enzyme, soluble cross-linked oligomers appeared initially. The opaque fibrin clots, produced by thrombin (pH 6.0) or contortrix procoagulant fraction (pH 7.3), were found to be devoid of alpha-polymers even after prolonged incubation with factor XIIIa. Our data suggest that the solubility and opacity of fibrin networks are not primarily related to the type of the cross-link (gamma-gamma versus alpha-alpha interactions).

Blood Coagulation↗
PubMed1976

[Structural model of factor VIII complex].

Gel filtration patterns on Sepharose CL-2B of functional subunits (procoagulant, ristocetin cofactor, antigen) derived from highly purified human factor VIII indicate that the native, intact complex is reproducibly eluted in the void volume. The complex is, however, degraded by proteolytic (and possibly other) enzymes contaminating the purified material during dialysis at room temperature. Resulting degradation products were fractionated by gel filtration and characterized by SDS-polyacrylamide electrophoresis. Our results suggest that factor VIII consists of identical subunits (mol. wt. about 500 000) which might be linked together by hydrophobic bonds. Only native high molecular weight aggregates posses ristocetin cofactor activity.

Factor VIII↗
PubMed1976

Reactivity of fibrinogen crosslinking sites in the absence of thrombin.

The reactivity of fibrinogen crosslinking sites with thrombin-free, preactivated factor XIII (F. XIIIa) was investigated under different conditions such as increased ionic strength, presence of urea, protamine sulfate (PS) or of varying concentrations of monodansylcadaverine (MDC). Crosslinking and incorporation of MDC into fibrinogen or fibrin gamma- and alpha-chains were evaluated by SDS-polyacrylamide gel electrophoresis. According to our results, rates of crosslinking of, and of MDC incorporation into, both gamma- and alpha-chains of fibrinogen were low under physiological conditions; they were not significantly influenced by the presence of either 1.0 M NaCl or 1.0 M urea. In contrast, 0.01% PS precipitated fibrinogen, and, simultaneously, significantly increased the rates of crosslinking and of MDC incorporation into both gamma- and alpha-chains. MDC, at concentrations above approximately 6 mM, also precipitated fibrinogen, and, up to a concentration of about 9 mM, markedly enhanced the reactivity of acceptor crosslinking sites. Our results suggest that solubility of fibrinogen and the conformational arrangement of its subunit chains are closely interdependent; the reactivity of crosslinking sites with F. XIIIa seems to be a function of this conformational state.

Animals↗
PubMed1976

Importance of protease inhibition in studies on purified factor VIII (antihaemophilic factor).

According to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW approximately 2 X 10(6) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37 degrees C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macromolecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

Antigens↗
PubMed1976

Auto-oxidation of hemoglobin in plasma.

The rate of auto-oxidation of human hemoglobin to methemoglobin was measured in plasma at 37 degrees C. Half-lives of hemoglobin were found to be 20 h, 12 h and 7 h at the oxygen tensions of 126, 57 and 23 mm Hg, respectively.

Hemoglobins↗
PubMed1975

[The fate of hemoglobin in the plasma].

The following aspects of intravascular hemolysis are briefly reviewed: pathogenesis; binding of hemoglobin to haptoglobin, hemopexin or serum albumin; hemoglobin oxidation, dissociation and renal excretion. The dissociation constant of hemoglobin tetramer into dimers was determined by gel filtration at 37 degrees C, using normal plasma for elution. This constant (5.1 x 10(-6) mol/l was used for calculation of renal clearance of hemoglobin. The rate of hemoglobin autooxidation was measured in normal plasma applying varying partial oxygen pressures. The results confirm that the formation of methemoglobin is enhanced under hypoxic conditions.

Erythrocyte Aging↗
PubMed1975

Plasmic degradation of human fibrinogen. III. Molecular model of the plasmin-resistant disulfide knot in monomeric fragment D.

A mixture of fragments D, derived from fibrinogen by plasmic degradation, was S-reduced and carboxymethylated. Individual chains were separated by gel filtration on Sephadex G-100 and characterized by peptide mapping, N-terminal amino acid analysis, polyacrylamide electrophoresis in sodium dodecyl sulfate, and amino acid composition. It was demonstrated that all D species contain the same alpha- and beta-chain remnants, having mol. wts of 10 000 and 45 000, respectively. Their heterogeneity was shown to be caused by the gradual degradation of the gamma-chain at its C-terminal end. Denatured fragment D was further degraded with plasmin in the presence of 2 M urea. One beta- (mol. wt 17 000) and two gamma-fragments (mol. wts 5000 and 6000) were split from fragment D, in addition to non-characterized small peptides, leaving behind a plasmin-resistant core, designated as fragment d. Fragment d was in turn reduced and carboxymethylated, and the resulting constituent chains were isolated by chromatography on carboxymethyl-cellulose and Sephadex G-100. The reduced alpha-, beta- and gamma-chain remnants of fragment d were found to have been derived from the N-terminal portion of fragment D and have estimated mol. wts of 9000, 24 000 and 13 000, respectively. A tentative scheme for the conversion of an early fragment D into the core fragment d is proposed. Our results conclusively support the model of asymmetric degradation of fibrinogen, according to which 2 mol of monomeric fragment D are produced from 1 mol of fibrinogen.

Amino Acid Sequence↗
PubMed1975

Plasmic degradation of human fibrinogen. IV. Identification of subunit chain remnants in fragment Y.

A method is presented for detection of cross-linking acceptor sites on fibrinogen chains, using monodansyl-cadaverine labeling in the presence of activated fibrin stabilizing factor, and polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Fluorescent gamma-chain monomers and dimers were produced at a considerably faster rate than the labeled alpha-chain derivative. Purified fragments X, Y and D were prepared all from the same plasmic digest of fibrinogen. Following incubation with fibrin stabilizing factor, thrombin and monodansyl-cadaverine, they were reduced with beta-mercaptoethanol and examined by sodium dodecyl sulfate/acrylamide electrophoresis. Three gamma-chains (mol. wts 49 000, 42 000 and 39 000) had reacted with dansyl-cadaverine while no alpha-chain remnant took up the label. Additional protein and carbohydrate staining further facilitated identification of the individual subunit chains. At least three critical peptide bonds, located on alpha, beta- and gamma-chain remnants, must be broken during conversion of fragment Y into D and E. Sequential cleavage results in heterogeneous appearance of reduced subunit chains. As a consequence, there exist several molecular entities of fragment Y, all of which may have the same molecular weight though they represent various products of progressive plasmic digestion. Our results are compatible with the model of asymmetric degradation of fibrinogen, according to which fragment X produces 1 mol of fragment E e and 2 mol of the monomeric fragment D.

Amino Acid Sequence↗