Assay of Fletcher factor (plasma prekallikrein) using an artificial clotting reagent and a modified chromogenic assay.

An artificial clotting reagent lacking in Fletcher factor (plasma prekallikrein, PPK) was made by mixing human plasma, activated by 5 mg/ml of celite, then kept 16 hours at 37 degrees to destroy most of the plasma kallikrein, plus rabbit plasma (which is devoid of XII and Fletcher activity). Chromogenic assay using a tripeptide substrate was also modified to exclude the interference of the endogenous contact factors. Celite eluate was used instead of kaolin or dextran sulphate for the activation. Using both these methods, it is possible to distinguish between Fletcher trait (PPK deficiency) and other contact factors such as factor XII and HMWK deficiencies, which do not activate with kaolin or dextran sulphate. These simple clotting and enzymatic assays give specific and well correlated results for PPK estimation.

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.

In vivo attempt to consume antithrombin III by i.v. injection of a thrombin-heparin mixture.

15 rabbits and 6 dogs were injected with a mixture of thrombin and heparin. Increasing amounts of thrombin, and various ratio of thrombin in relation to heparin, were used. It was found that huge amounts of thrombin could be perfused, under the protection of heparin, without untoward effects. But the high amount of thrombin needed to obtain a 50% reduction of the circulating AT III required a corresponding high amount of protective heparin. Since the secondary injection of protamin sulphate (after the end of the perfusion with the thrombin-heparin mixture) was precipitating the DIC syndrome, such attempts to decrease AT III in man are not feasible.

[New preparation method of C1 esterase for the dosage of its plasma inhibitor]. / Nouveau mode de préparation de la c1 estérase pour le dosage de son inhibiteur plasmatique.

A purification method for C1 esterase is described. The final product significantly improved the sensitivity and the specificity of the enzymatic measurement of its plasma inhibitor C1-INH or alpha 2-neuraminoglycoprotein (alpha 2-NGP) by esterolysis of a synthetic substrate N-acetyl-L-tyrosine ethyl ester (ALTEe). A comparative study was done between the chromatographed C1 esterase and the native serum euglobulins: qualitative and quantitative determination of the serum contaminants, enzymatic activity measurement of C1-INH in normal subjects and in patients suffering from hereditary angioneurotic oedema (OANH) as well as in therapeutical C1-inhibitor concentrates.

Attempt to prevent hepatitis by using specific anti-HBs immunoglobulin.

Sixteen batches of specific anti-HBs immunoglobulin have been prepared from December 1970 up to now. They were used in an attempt to prevent hepatitis B: (1) By a single injection, within seven days, in patients exposed by HBs Ag-positive blood transfusion: with a dose of 0.16 ml/kg, two hepatitis B cases were observed among 29 followed up patients out of 43 cases; with a dose of 0.5 ml/kg, no hepatitis was reported to us in the next 26 patients. (2) By a single injection, within seven days, in laboratory and hospital personnel accidentally contaminated. With a dose of 0.08 ml/kg administered to more than 1000 persons, an excellent protection was observed. (3) By injections repeated every five weeks during the first four months, then more widely spaced during the following months, in hemodialysis units staff. The attack rate of clinical hepatitis B was reduced from about 44 per cent to 0 per cent. No hepatitis case was observed in 90 persons protected from four to 26 months. In a control group (eight subjects) three hepatitis B cases occurred (37 per cent).

[A new method of functional assay of des-gamma-carboxyprothrombin using staphylocoagulase. Application to the diagnosis of hepatocellular carcinoma]. / Nouvelle méthode de dosage fonctionnel de la dé-gamma-carboxyprothrombine à l'aide de staphylocoagulase. Application au diagnostic des carcinomes hépatocellulaires.

A new method to assay des-gamma-carboxyprothrombin (DCP) activity is described, using staphylocoagulase on undiluted citrated plasma after adsorption with bentonite (to remove fibrinogen), then with aluminium hydroxide. The thrombin-coagulase which is formed is measured by following the increase in optical density per minute of a chromogenic substrate. The results are expressed in milliunits (m.U.). The new method is sensitive and specific. It confirms the usefulness of the DCP assay for the diagnosis of hepatocellular carcinoma (Liebman et al.). Ninety-six normal subjects had levels of DCP ranging from 0 to 12 m.U. Out of 42 patients with hepatocarcinoma, 30 (71%) had DCP levels higher than 15 m.U. The increased DCP appears to be complementary to alpha-foetoprotein, since one or the other marker were positive in 37 out of 40 cases (92.5%) of hepatocellular carcinoma. Chronic hepatitis or cirrhosis (36 cases) and non-hepatocellular liver cancer (9 cases) gave normal DCP values.

[Parvovirus B 19]. / Le parvovirus B 19.

Since 1981, many reports have contributed to establish that a human parvovirus (parvovirus B 19), which had been isolated only in asymptomatic blood donors, is the causative agent of transient but intense erythroblastopenia in patients with different types of chronic haemolytic anaemia. In vitro cultures of erythroid inhibitors revealed an inhibition by parvovirus B 19, abolished by convalescent serum. In healthy subjects without chronic haemolysis, first contact with parvovirus B 19 results in an inconstant influenza-like syndrome with transient erythroblastopenia which does not produce symptoms since the normal erythrocyte life span covers the effect of parvovirus B 19 on bone marrow. Parvovirus B 19 is also suspected to be the causative agent of erythema infectiosum (fifth disease) which occurs in children.

Contact activation of prekallikrein and of fibrinolysis by heparinoids and heparin.

Contact activation of plasma prekallikrein (PPK) assayed by a tripeptide chromogenic substrate, and surface-mediated fibrinolysis by the euglobulin test (both tests being insensitive to the anticoagulant effect of heparin and heparinoids) were studied. The following substances were tested: Dextran solutions of different molecular weight (M.W.), heparinoids such as dextran sulphate, pentosan and mannuronate sulphuric polyesters. Liquoid and Moranyl and various commercial therapeutic preparations of heparin. Most of these anionic polyesters are able to activate both PPK and fibrinolysis, dextran sulphate (500,000 M.W.) being the most active. Factor XII, PPK and HMWK are necessary for full activation. Besides dextran sulphate, other heparinoids such as Na pentosan polysulphate, or Na polyanhydromannuronic sulphuric acid, are also contact activators. This may explain some of their side effects observed in vivo. Non-sulphated dextrans used in therapy have no activating effect. Nine therapeutic commercial preparations of heparin were tested. They appear to have a slight activating effect on PPK but an uncertain effect on fibrinolysis. Such factor XII activability has until now been unnoticed, being masked by the anticoagulant activity of heparinoids and heparins on later phases of blood coagulation. It appears that heparin is unable to prevent contact activation in vivo as well as in vitro. This allows PPK assays during extracorporeal circulation in the presence of circulating heparin.