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Biomedical subjects

M C Boffa

Publications and source records attributed to M C Boffa.

90 records · Page 5Linked to original sources
PubMed1979

Purification and properties of prothrombin modified by asbestos filtration of human plasma.

Filtration through asbestos filter (Seitz) of human plasma modified the prothrombin molecule as previously shown. Factor II could no longer be activated by physiological activators (Ca++ + phospholipid + V and Xa) but reacted readily with staphylocoagulase. The separation and purification of the modified prothrombin allowed allowed the preparation of two fractions: A small slightly modified accessory fraction, "prothrombin-asbestos-1", lost its ability to be activated by physiological activators, and its ability to be adsorbed by barium citrate, but retained the immunological reactivity of fragment 1, as well as the mobility and molecular weight of unmodified prothrombin. A main fraction, "prothrombin-asbestos-2" appeared to be a modified prothrombin which has lost its N-terminal extremity. It was not adsorbed by barium citrate and could not be activated by physiological activators. It possessed a reduced electrophoretic mobility, as well as a reduced molecular weight (39,000), which are properties similar to those of thrombin. Both fractions 1 and 2 were devoid of thrombin activity. Asbestos was thus able to break the prothrombin molecule non enzymatically, the amputation of the N terminal extremity being responsible for the new functional and physicochemical properties of the molecule. Staphylocoagulase appeared not to need the N terminal extremity of the molecule of prothrombin to form the active thrombin-coagulase complex.

Asbestos↗
PubMed1977

Properties of heparin--poly(methyl methacrylate) copolymers. II.

A heparin--poly(methyl methacrylate) copolymer in which heparin has been covalently bound was studied. This copolymer showed no release of heparin but presented heparin-like anticoagulant activity when suspended in plasma. After contact with plasma, the copolymer lost part of its antithrombin activity which could be restored by a high ionic-strength medium. Because of its solid form, this copolymer offers new possibilities for producing interesting anticoagulant surfaces.

Anticoagulants↗
PubMed1977

Polytetrafluoroethylene-N-vinylpyrrolidone graft copolymers: affinity with plasma proteins.

N-vinylpyrrolidone was grafted onto films, grains, and tubes of polytetrafluoroethylene (PTFE) using a radiation procedure. By controlling the experimental conditions, it was possible to obtain an analogous grafting percentage on films and grains. Kinetic grafting onto a tube might be used under certain conditions to preserve both size and water tightness. Poly(vinylpyrrolidone) (PVP)-grafted material became hydrophilic and slightly swollen according to the grafting percentage obtained. A scanning electron microscope (SEM) analysis of polymer surfaces showed preferential polymerization sites at a low radiation dose. A regular, velvet-like surface was obtained for a high grafting percentage induced at a low radiation intensity. Biological properties of PTFE were compared before and after PVP grafting. PTFE appeared to be a nonreactive polymer with respect to the blood-clotting proteins. No significant change was observed using different granulometric sizes of PTFE grains, and its noteworthy inertness was preserved after PVP grafting. Interactions between polymer surfaces and human plasma proteins were analyzed using 125I-isolated proteins. Fibrinogen and immunoglobulins have a high affinity for PTFE; on the other hand, albumin was weakly adsorbed on the polymer surface. Marked changes in protein adsorption were observed after PVP grafting. Hydrophilic group grafting induced an increase of albumin adsorption and a decrease of fibrinogen adsorption. Prothrombin affinity was not significatly affected by modified PTFE. Studies of serum protein adsorption using solid-phase radioimmunoassays were developed with monospecific and polyvalent antisera. After incubation of the whole serum protein and polymer, immunoglobulins were found to exhibit the major part of bound protein radioactivity on the polymer surface. No evidence of increasing adsorption of immunoglobulins from rabbit serum after immunization by PTFE or PTFE/PVP was observed. The immunoglobulin adsorption was submitted to a competitive effect with the other serum proteins. It was possible to postulate the competitive action of albumin, particularly after biological property changes induced by chemical grafting.

Animals↗
PubMed1976

A phospholipase A2 with anticoagulant activity. I. Isolation from Vipera berus venom and properties.

An anticoagulant protein has been isolated by DEAE cellulose chromatography and gel filtration from the venom of the Vipera berus orientale (Eastern Europe). Purification has been completed by elution on carboxymethyl cellulose with continuous gradient at constant pH. The inhibitor of coagulation was separated from the other venom enzymes, e.g. procoagulant, fibrinogenolytic, aminoesterase and amino acid oxidase activities. It was also separated from other phospholipase components which were not related to the anticoagulant property. The inhibitor appeared as a simgle polypeptidic chain protein, formed by 119 amino acid residues, with a molecular weight of 13400 and an isoelectric point of 9.2. At low saline molarity, a monomer-trimer transition of this protein was observed. Both forms had the same amino acid composition. There were six disulfide bridges without free SH groups per phospholipase molecule. Deprived of any proteolytic activity, the clotting inhibitor displayed a high phospholipase activity in the presence of calcium. Activity did no appear with EDTA buffer deprived of cation. Finely dispersed micellar suspensions were found suitable for obtaining the highest phospholipase activity. High sodium cholate concentration or methanol/chloroform/ether solvent were effective without loss of enzymatic activity. As characteristis of phospholipase A2 (EC 3.1.1.4), the degradation products identified on thin-layer chromatography induced hemolysis of human erythrocytes. The apparent Km value 1.25 - 10(-3) M was determined on phosphatidylcholine isolated from ovolecithin. This purified berus inhibitor would be of value for investigating the involvement of phospholipids in the clotting mechanism.

Amino Acids↗
PubMed1976

A phospholipase A2 with anticoagulant activity. II. Inhibition of the phospholiped activity in coagulation.

An anticoagulant factor with phospholipase A2 activity has been isolated from Vipera berus venom. Phospholipase activity was studied on platelet phospholipid and on brain cephalin. The venom factor showed a potent anticoagulant activity: 1 mug impaired the clotting of 1 ml of citrated recalcified platelet-poor plasma. The anticoagulant inhibited clotting by antagonism to phospholipid. The antagonism constant (Kan = 6.8-10(-9) M) demonstrated the high affinity of the inhibitor for phospholipid. As with other phospholipases A2, the venom factor was thermoresistant but very sensitive to photo-oxidation. Both activities (anticoagulant activity and phospholipase activity) were not markedly dissociated by either denaturation or neutralization processes. Slightly different curves of photo-oxidative inactivation of both activities suggested the presence, on the molecule, of two very close sites responsible for phospholipase and anticoagulant activities. The inhibitor effect on coagulation was independent of the hydrolysis process. In fact, lysoderivatives and fatty acids, resulting from complete hydrolysis with the venom factor, were as active as the native phospholipids. Moreover phospholipase A2 from other viperidae venom, which did not have anticoagulant activity, produced similarly active lysoderivatives. This showed that the cleavage of the beta-acyl bond does not interfere with the activity of phospholipid. A possible mechanism of clotting inhibition by the venom factor was proposed. Owing to its high affinity for phospholipid, the inhibitor would complex phospholipid at its protein binding site impairing the normal arrangement of coagulation protein factors and, consequently, their activation. The positive charges of the inhibitor (pI = 9.2) could bind with phosphoryl or carboxyl groups of phospholipid, making them unavailable for protein binding. The complex formation involves a loss of dissociating capacity of the enzyme towards its substrate. This required an additional interaction of the inhibitor with a coagulation protein factor. The inhibitor could be removed from the complex by specific antibodies, permitting recovery of normal phospholipid-protein interaction. The role of calcium in the complex has not yet been elucidated. This venom factor affords a useful tool for investigating the phospholipid-clotting protein interaction.

Anticoagulants↗
PubMedDate not supplied

Antiphosphatidylethanolamine antibody as the sole antiphospholipid antibody in systemic lupus erythematosus with thrombosis.

A previously healthy 34-year-old woman, was diagnosed as having systemic lupus erythematosus (SLE), with membranous glomerulopathy which improved rapidly. Neither lupus anticoagulant nor anticardiolipin antibodies were detected in her plasma. After three months of total remission, she developed a severe pulmonary thromboembolism for which no specific biological cause was found. Her plasma was analysed for different antiphospholipid antibodies: lupus anticoagulant and anticardiolipin antibodies were again negative. Using an ELISA prepared with either five different anionic phospholipids or zwitterionic phosphatidylethanolamine, solely an anti-phosphatidylethanolamine IgG was discovered in her plasma. In lupus patients, the presence of antiphospholipid antibodies is now well recognized as a high risk factor for repeated thrombosis and/or recurrent abortions. This case suggests that the presence of antiphosphatidylethanolamine antibody should be investigated in cases of unexplained thrombosis in SLE, where the usual clinical and biological investigations have failed to shed light.

Adult↗