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.

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.

Effects of conditioned medium from bone marrow of normal and Ara-C-treated mice on hemoglobin synthesis.

Differentiation of CFUs toward erythropoiesis has been demonstrated in mice treated with a single dose of Ara-C, and this preferential differentiation was shown to be under the influence of diffusible factors. The aim of the work reported here was to determine whether these factors might include erythropoietin (Ep). This possibility was explored by testing the effect of the Ara-C-induced factor(s) on hemoglobin synthesis, and the effects of anti-mouse Ep on the activity of Ara-C-induced factors. When the late erythropoiesis stage of fetal liver cells was stimulated by bone-marrow-conditioned medium, no significant difference was observed between the effects of conditioned media obtained from normal versus Ara-C-treated bone marrow cells. Bone-marrow-conditioned medium induced a dose-dependent effect on hemoglobin synthesis by fetal liver cells (or adult bone marrow cells); the resulting curve was not parallel to the log-dose-response curve of purified mouse Ep. Anti-mouse Ep inhibited the activity of mouse Ep in fetal erythroblast cultures, but did not affect the stimulatory activity of conditioned media. Below the plateau doses no cumulative erythropoietic effect was observed in fetal erythroblast cultures when conditioned medium was added to Ep. Thus the present data suggest that the effect of bone-marrow-conditioned medium results from at least two independent factors: the first one active on CFUs differentiation towards erythropoiesis, and the second one active at a later stage of erythroid maturation. These factors can be considered to be molecules with an immunogenic structure unrelated to that of Ep.

Interactions between human plasma proteins and heparin-poly(methyl methacrylate) copolymer.

A solid Heparin-PMMA copolymer has been synthetized by a radical polymerization of methyl methacrylate from oxidative reaction initiated by Ce4+ ions in the presence of heparin. Covalently linked heparin was 10% of copolymer weight. The antithrombin activity of the copolymer corresponded to 1% of grafted heparin. PMMA sequence of the copolymer played the leading role in fibrinogen, immunoglobulins, transferrin and albumin adsorption. These proteins adsorbed on the copolymer, showed different competitive desorption pattern in the presence of whole plasma: fibrinogen presented the highest degree of affinity for the copolymer. The heparin part of the copolymer was responsible for antithrombin III adsorption and for decrease of factor V activity. Active antithrombin III was eluted. An inactivation of factor V in plasma was observed using high concentrations of soluble heparin. This result suggested that copolymer heparin chains, even devoid of antithrombin activity were involved in this inactivation. With Heparin-PMMA copolymer, plasma clotting pro-enzymes behaved differently than on heparin-sepharose copolymer:disappearance of factor XI activity, decrease in prekallikrein activity and activation of factor IX were observed. PMMA sequences were responsible for factor IX activation.

Pachydermoperiostosis with gastric hypertrophy, anemia, and increased serum bone Gla-protein levels.

We evaluated a patient in whom pachydermoperiostosis occurred in conjunction with anemia and gastric hypertrophy. The mechanism of the anemia appears multifactorial because, besides a myelofibrosis, a serum inhibitor of the late stage of erythropoiesis was detected. The elevated serum bone Gla-protein (osteocalcin) favors the hypothesis that primary hypertrophic osteoarthropathy represents an imbalance between increased osteoblastic bone formation and normal bone resorption.

Preparation and biological properties of monoclonal antibodies against human erythropoietin.

Several monoclonal antibodies (Mabs) against human erythropoietin (H-EPO) were obtained by fusion of myeloma cells with splenocytes from mice immunized with purified recombinant H-EPO. Three Mabs (D7, E14 and E73) were selected by radioimmunoprecipitation on the basis of their high affinity against H-EPO. Their dissociation constants were 0.3 nM for E14 and D7, and 17 nM for E73. The immunochemical properties of these Mabs were analyzed in respect to their capacity to react, to purify, and to inhibit the biological activities of H-EPO. a) detection by Western blotting techniques: among the 3 Mabs only D7 was effective by this technique. b) purification: the best results were observed with E14, an approximately 200-fold purification of H-EPO from culture supernatants was obtained in a single immunopurification step. c) inhibition of the biological activity: the specific binding of 125I-labelled H-EPO to its cellular receptor was inhibited by E14 and E73 but not by D7. These three Mabs exhibited similar effects as far as the inhibition of the proliferation of H-EPO dependent cells (measured by 3H-thymidine incorporation) was concerned.

[Secondary polycythemia with hypererythropoietinemia. Supratentorial meningioma]. / Polyglobulie secondaire avec hyper-érythropoiétinémie. Méningiome sus-tentoriel.

A meningioma was discovered in a 66 year old man with non-progressive alcoholic cirrhosis and abstinence for the 5 previous years. The development of polycythemia ten months later was related to an inappropriate secretion of erythropoietin. Conventional causes for this phenomenon could not be found. Erythropoietin assays in erythroblast cultures from both the patient's blood and post-mortem, from tumor fragments demonstrated a significant elevation of erythropoietic activity. The diagnosis of polycythemia secondary to meningioma is suggested. Only one similar case has apparently been reported.

[Polycythemia secondary to hepatic hemangioma. Demonstration of tumoral erythropoietin secretion]. / Association polyglobulie angiome hépatique. Mise en évidence d'une activité "érythropoïétine-like" intratumorale.

We report the case of a 37-year old man with polycythemia related to hepatic tumor with elevated serum erythropoietin concentrations. Left hepatic lobectomy was performed 20 months after admission. Microscopic examination revealed a typical hemangioma. Erythropoietin in tumoral extract was elevated. Two months later, the hematocrit and the serum erythropoietin level were within normal range. To our know-ledge, this is the first report in literature.

[Secondary paraneoplastic polycythemia of testicular cancer]. / Polyglobulie secondaire paranéoplasique d'un cancer testiculaire.

The authors report the case of a 41-year old patient with polycythaemia and a mixed testicular tumour (seminoma with choriocarcinomatous contingent). The demonstration of a raised erythropoietin level in the tumor tissue with a high gradient in relation to the serum argues in favour of secondary paraneoplastic polycythaemia. With a follow-up of 5 years, the patient is still in complete remission from his two diseases. This case may represent the first case of testicular tumour responsible for secondary polycythaemia.

Inhibitory and anti-inhibitory factors of rat serum active on the G1-S transition of hepatocyte cell cycle.

Rat hepatocytes are responsive to a serum factor inhibiting their progression through the cell cycle from the late G1 phase to the S phase. After fractionation of normal adult rat serum by two chromatographic steps on DEAE cellulose and sephadex gel filtration, the inhibitory activity was linked to proteins having a high electronegative charge and of apparent high molecular weight. Polyacrylamide gel electrophoretic analysis of active fraction showed that the alpha1 macroglobulin was its main component. Male and female baby rats were sensitive to the inhibitory factor from normal rats. Contrary to the normal adult rat serum the whole hepatectomized adult rat serum did not exhibit any ingibitory activity on the G1-S transition. However, two components having antagonist activities: an alpha1 globulin and a gamma globulin, were separated by chromatographic procedures from hepatectomized rat serum. (a) The alpha1 globulin showed an inhibitory activity. It had an apparent molecular weight lower than that found in normal rats. Its activity was sex related: only male baby rats were responsive. (b) The factor present in the gamma globulin fraction was found to be antagonistic to the alpha1 globulin factor. Its occurrence after hepatectomy explains the absence of inhibitory activity in the serum of hepatectomized rats.

Serum-free culture of primitive murine hemopoietic colony-forming cells.

We report the effect of four sources of hemopoietic growth factors, alone or in combination, on colony growth in serum-free cultures of bone marrow from normal mice or marrow from mice pre-treated with 5-fluorouracil (5-FU-bm). The four supplements were: mouse spleen conditioned medium (SCM, a source of multi-lineage colony-stimulating activity, multi-CSA), human placental conditioned medium (HPCM, a source of synergistic activity), pregnant mouse uterus extract (PMUE, a source of M-CSA) and erythropoietin (Epo). First, in cultures of normal marrow, only PMUE and SCM induced significant colony growth when added alone. The majority of those colonies contained granulocytes and macrophages (myeloid colonies). In Epo-supplemented cultures, only SCM supported the growth of erythroid bursts and mixed erythroid-myeloid colonies. HPCM thus appears to be a poor source of multi-CSA. Second, in cultures of 5-FU-bm, few colonies developed if any of the above supplements were added alone. Only SCM + Epo together stimulated the formation of a low number of very large, mixed erythroid/myeloid/megakaryocyte colonies. HPCM, but not SCM, synergized with PMUE to augment myeloid colony numbers. Hence, SCM appears to be a poor source of synergistic activity (SA). In cultures of 5-FU-bm already supplemented with HPCM + PMUE, the addition of Epo did not change total colony numbers but did induce erythroid differentiation in one third of the colonies present. These data suggest that multi-CSA and SA may be expressed by different factors and that 5-FU pre-treated marrow contains: a population of primitive multipotential progenitors which form large, mixed colonies in the presence of SCM + Epo, and a larger Epo-sensitive population which also requires HPCM + PMUE to form mixed colonies.

[Polycythaemia and uterine fibroma. A case with in vitro demonstration of an erythropoietic activity in the tumor (author's transl)]. / Polyglobulie et fibrome utérin. Mise en évidence in vitro d'une activité érythropoïétique dans l'extrait tumoral.

A new case of polycythaemia associated with an uterine fibroma is presented. The presence of erythropoietin in the tumor has been demonstrated by an in vitro technique of titration using a microculture of foetal mouse liver cells. The study of 46 previously reported cases show that the usual pattern is that of an isolated polycythaemia occurring in a woman in her fifty and of a large fibroma. The surgical cure of the tumor relieves rapidly and definitively the polycythaemia. The hypothesis of an inappropriate secretion of erythropoietin seems admitted, three factors playing probably a role in the occurrence of the polycythaemia: size of the fibroma, state of the iron stores and presence of an inhibitory factor against erythropoiesis.

[Inhibitory effect of human alpha 2-macroglobulin and a peptide released by trypsin, on the G1-S transition of hepatocytes in vivo]. / Effet inhibiteur de l'alpha 2 macroglobuline humaine et d'un peptide libéré par la trypsine, sur la transition G1-S des hépatocytes, in vivo.

Highly purified human alpha 2 M inhibits hepatocyte proliferation. 1 mg of alpha 2 M corresponds to 1 baby rat unit (BRU). alpha 2 M is bound to a low molecular weight glycopeptide, which is released during trypsinization of alpha 2 M. 3 micrograms of trypsin-treated alpha 2 M release 1 BRU. alpha 2 M and the glycopeptide have been shown to be identical, respectively, to high and low molecular weight components present in normal human plasma. Both components inhibit the G1-S transition of the hepatocyte cycle. alpha 2 M acts as an antagonist to the inhibitory effect of the glycopeptide when the molar ratio of trypsin to alpha 2 M is greater than 2.

Rat serum factors inhibiting the G1-S transition in hepatocytes. I. Production of a low molecular weight inhibitor by proteases or liver fractions.

An attempt was made to detect the serum factors inhibiting the G1-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G1-S inhibitory factor. The same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10(3). The active period in the cell cycle of both the LMW and HMW factors was the same: the late G1 phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. An enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.

Complete replacement of serum in cultures of murine primitive erythroid and multipotential progenitor cells: absolute requirement for spleen conditioned medium.

We describe a serum-free medium for the formation of erythropoietic bursts by murine bone marrow cells. Iscove's modified Dulbecco's medium supplemented with bovine serum albumin, iron-saturated transferrin, soybean phospholipids and cholesterol supported burst formation. The further addition of hemin increased burst numbers to above those obtained in serum-containing cultures. With or without hemin, a source of burst-promoting activity (BPA) (crude or partially purified spleen conditioned medium) and erythropoietin were essential. This system provides a sensitive assay for BPA. Of all colonies developing in these cultures, 16% were pure erythroid, 17% mixed erythroid/myeloid, 36% macrophage, 19% macrophage/basophil and macrophage/neutrophil, 9% basophil and 2% neutrophil.