Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin.

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.

Albumin, fibrinogen, prothrombin and antithrombin III variations in blood, urines and liver in rat nephrotic syndrome (Heymann nephritis).

Albumin, fibrinogen, prothrombin and antithrombin III (AT III) variations have been studied in blood, urines and liver during an experimental nephrotic syndrome in rats (Heymann nephritis). A quantitative morphometric study (light microscopy) has been performed in the liver using an immunocytochemical technique--(PAP) method--to evaluate the protein synthesis by the number of protein-containing hepatocytes. Some sections were also studied by electron microscopy. The nephrotic animals were compared with control rats. In the blood of nephrotic rats, fibrinogen and prothrombin concentrations were increased and albumin and AT III concentrations were decreased. In the urines of nephrotic rats, albumin, prothrombin and AT III were lost, but no fibrinogen. The morphometric study in the liver has shown a significantly higher number of fibrinogen and prothrombin-containing hepatocytes in nephrotic rats than in controls, suggesting an increased synthesis of these proteins; no change was observed concerning albumin and AT III between nephrotic and control animals. In electron microscopy, albumin was demonstrated in Golgi apparatus, proving that the peroxidase-positive cells are related to protein synthesis. These results show that the mechanisms of regulation of the protein synthesis during nephrotic syndrome are different from one protein to another and, particularly, that their blood level is not the only regulating factor for their synthesis.

Fibrinogen Bondy: a new case of dysfibrinogenemia. Isolation of the abnormal fibrinogen molecules.

In a 81 year old health woman, gross abnormalities of fibrin formation led to the discovery of an abnormal fibrinogen named fibrinogen Bondy. Clottability of purified fibrinogen Bondy was only 53% compared to 95-98% for normal fibrinogen. Functional studies revealed (i) delayed coagulation by thrombin and batroxobin (Reptilase), (ii) incomplete release of fibrino-peptides A and B, (iii) poor fibrin monomer aggregation, (iv) delayed fibrin proteolysis by plasmin. Electrophoretic mobility of fibrinogen Bondy, its three chains and the products of fibrin cross-linking, was normal. Fibrinogen NH2-terminal residues of fibrinogen Bondy were found to be normal. The presence of Ala, in addition to Gly and Tyr in the fibrin clot and its supernatant, showed that a part of fibrinogen molecules was not clotted, i.e. either copolymerised with fibrin or remaining in solutions. Gel filtration of the supernatant allowed the separation of both soluble complexes and fibrinogen. This fibrinogen population was shown to be unclottable by thrombin and to inhibit clotting of normal fibrinogen.

A new variant of human prothrombin: prothrombin Metz, demonstration in a family showing double heterozygosity for congenital hypoprothrombinemia and dysprothrombinemia.

Investigation of a mild hemorrhagic tendency in a French family revealed the father to be heterozygous for hypoprothrombinemia while the mother was heterozygous for dysprothrombinemia. All possible genetic combinations could be demonstrated. Among the children the double heterozygosity encountered in 3 of them allowed us to discover an abnormal prothrombin, prothrombin Metz, which generates an abnormal thrombin less sensitive to inactivation by antithrombin III than normal thrombin.

Respective roles of antithrombin III and alpha 2 macroglobulin in thrombin inactivation.

In order to investigate the mechanism of thrombin inactivation in the presence of both antithrombin III (AT III) and alpha 2-macroglobulin (alpha 2 M), thrombin and the inhibitors have been purified from human material and thrombin inactivation studied using purified reagents either alone or added to defibrinated plasma. Comparison of clotting and amidolytic activities of residual thrombin allowed to measure the amount of thrombin bound to alpha 2 M. In a purified reagent system as well as in plasma, part of exogenous thrombin is bound to alpha 2 M. The amount of bound thrombin is related to alpha 2 M concentration. Conversely, previous plasma alpha 2 M depletion by immunoabsorption increases the consumption of heparin-cofactor activity by exogenous thrombi. Thus AT III and alpha 2 M compete for thrombin inactivation. This finding could be of practical interest in clinical situations associating high plasma alpha 2 M levels and a decrease of AT III concentrations.

Antithrombin III versus prothrombin in liver cirrhosis.

In patients with liver cirrhosis, a close relationship was observed between the respective levels of antithrombin III and prothrombin, both below 50% in 20 our 27 patients. The absence of any thrombotic complication, despite low plasma antithrombin III, suggests that the preserved balance between the inhibitor and the zymogens of the inhibited enzymes could have a protective effect against thrombotic tendency.

[Severe blood coagulation disorder as the first sign of a peritoneal hemangio-endothelioma: a new therapeutic approach (author's transl)]. / Perturbations sévères de l'hémostase révélant abord thérapeutique.

The authors describe a 3 1/2 month old infant with hemangio-endothelioma and a severe blood coagulation disorder. The tumor was inoperable and the severe blood coagulation disorder posed considerable therapeutic problem. After treatment with massive amounts of blood clotting factors, an antifibrinolytic drug and radiotherapy, the child's condition improved.

[Anticoagulants with antithrombinase activity in systemic lupus erythematosus (author's transl)]. / Anticoagulants circulants d'activité antiprothrombinase au cours des néphropathies lupiques: incidence et signification pronostique.

An anticoagulant with antithrombinase activity was looked for in 49 patients with systemic lupus erythematosus and found to be present in 14 of them. The latter were suffering from severe and progressive lupus, specially with arterial involvement. The presence of an isolated antithrombinase does not contraindicate a renal biopsy provided the platelet count and other coagulation factors are normal.

[Familial thromboembolic disease associated with antithrombin III deficiency (author's transl)]. / Maladie thrombo-embolique familiale et déficit en antithrombine III.

The authors report the observation of a new kindred with hereditary antithrombin III deficiency. In the last three generations, the family comprised 10 subjects, 7 of whom were affected: the grandmother had recurrent thrombophlebitis; her three sons died from pulmonary embolism at 22, 26 and 28 respectively and her daughter had repeated bouts of thrombophlebitis. In patients with hereditary antithrombin III deficiency, venous thrombosis occurs under similar conditions as, and is clinically similar to, thrombosis in patients without this defect. Usual tests of hemostasis are normal. The diagnosis is however suspected through an history of recurrent episodes and of similar cases in relatives. The diagnosis is confirmed by demonstration of low levels of antithrombin III in suspected patient and family. The disease is transmitted as autosomic dominant trait. Heparin is ineffective but oral anticoagulants may prevent occurence or recurrence of thrombosis in patients with this genetic defect.

[Nephrotic syndrome, antithrombin III deficiency and recurrent thrombosis. The value of vitamin K antagonists]. / Syndrome néphrotique, déficit en antithrombine III et thromboses à répétition. Intérêt des antagonistes de la vitamine K.

In a 7 year-old girl presenting with nephrotic syndrome and repeated episodes of thrombosis, a decrease in antithrombin III and in vitro inactivity of heparin were observed. Treatment by vitamin K antagonists prevented further thromboembolic episodes.

[Initiation in vivo of blood coagulation. The role of white blood cells and tissue factor (author's transl)]. / Déclenchement in vivo de la coagulation. Rôle des leucocytes et du facteur tissulaire.

Tissue factor is an ubiquitous phospholipid-protein complex, which triggers blood coagulation through the so-called extrinsic pathway. Reactions initiated by tissue factor bypass many of the early stages of coagulation (contact phase) and involve factors VII, X, V, II and fibrinogen but also factor IX (and VIII) as it was recently demonstrated. So, it appears that tissue factor has a key-role in the haemostasic process as it has been suggested by the mildness or the absence of haemorrhagic syndrome in contact factors deficiencies. Tissue factor activity has been found in many types of cells, especially in white bloods cells. Experimental studies have demonstrated the presence of tissue factor activity in polymorphonuclears, lymphocytes, monocytes (or macrophages). This activity is enhanced by gram-negative endotoxin stimulation, inflammation, cell mediated immunologic phenomena or malignancy. These data are in good agreement with a wild range of features observed in human pathology: fibrin deposits in inflammatory lesions, disseminated intravascular coagulation (DIC) during the course of gram-negative septicemias or acute promyelocytic leukemias, local thrombi at the early phase of graft rejection. The protective effect of a phospholipase C against DIC induced in rats by tissue factor infusion suggests in the future, a specific therapy would be possible in man that, in the frequent clinical conditions involving clotting activation through tissue factor pathway.

[Heparin inhibition of the antithrombin activity of alpha-2-macroglobulin]. / Inhibition par l'héparine de l'activité antithrombique de l'alpha-2-macroglobuline.

The interaction between thrombin and alpha-2-macroglobulin was studied on human purified materials, either in the presence or in the absence of heparin, by kinetic analysis of thrombin inhibition and polyacrylamide gel electrophoresis. In the absence of heparin, binding of thrombin to alpha-2-macroglobulin, shown by electrophoresis, leads to the loss of the coagulant property of the enzyme. In the presence of heparin the rate of inhibition of thrombin clotting activity by alpha-2-macroglobulin is strongly decreased. Heparin binds to thrombin, impairing the formation of thrombin-alpha-2-macroglobulin complex. These data show that heparin paradoxically protects thrombin from inhibition by alpha-2-macroglobulin whereas it increases the enzyme inhibition by antithrombin III. Such a phenomenon could be of practical interest for treatment of thrombosis in patients with high plasma level of alpha-2-macroglobulin and low level of antithrombin III, such as occurs in the nephrotic syndrome.

[The system Hemalog D in paediatrics (author's transl)]. / Le système Hemalog D en pédiatrie.

The Hemalog D was studied at the Childrens' Hospital to examine its performance in paediatrics. All the subjects studied were children with non-hematologic diseases; both venous and capillary blood samples were taken. The differential was performed in parallel by the Hemalog D and the technicians of the Hematology Laboratory. There was an excellent correlation between the two methods for neutrophils and lymphocytes, a good correlation for eosinophils and a weaker correlation for monocytes and basophils. No difficulties were encountered during the course of the study. It must be noted, however that capillary samples caused a Low Rate alarm to be registered.

[Influence of the hematocrit on the formation of Heinz bodies within the red cells (author's transl)]. / Influence de l'hématocrite sur la formation provoquée de corps de Heinz intra-érythrocytaires.

The increased in vitro formation of Heinz bodies in erythrocytes exposed to oxidant agents (acetylphenylhydrazine) is an excellent screening test for the diagnosis of various congenital abnormalities of red blood cells (G-6-PD deficiency, unstable haemoglobins). This phenomenon is strongly dependent of the PCV of the test blood. Anemia by itself (hematocrit less than 30 p. cent) may be responsible for an increased formation of Heinz bodies even in the absence of any erythrocyte abnormality. This finding confirm that the results of the test are of value only if the PCV of blood samples is adjusted to 40 p. cent.