Effect of proteolysis on quantitation of plasma fibronectin concentration by two immunoassays (electroimmunoassay and immunoturbidimetric technique).

Quantitation of fibronectin (FN) concentration is strongly influenced by FN fragmentation with trypsin, kallikrein and plasmin. Digestion by trypsin and kallikrein leads to a progressive decline in FN detectability by the immunoturbidimetric technique to zero values but is associated with an increase in the height of rockets in the Laurell's electroimmunoassay. Plasmin mediated FN fragmentation induces a strong overestimation of the FN content by the electroimmunoassay and, at very high enzyme concentrations, provokes an underestimation of FN by the immunoturbidimetric technique. The decline in FN reactivity in the immunoturbidimetric assay coincides with the disappearance of heavy fractions migrating only slightly faster than native FN in SDS-PAGE. The increase in the height of rockets in the electroimmunoassay is the highest when fractions of intermediate rate of migration prevail in the SDS-PAGE pattern. Concomitant use of these two immunoassays can distinguish native FN from its degraded form and may possibly provide a partial explanation for discrepancies in published studies on the concentration of circulating FN in various pathological states.

Separation of fibrinogen degradation products (FDP) by means of adsorption to insolubilized fibrinmonomer (FM-ag).

The phenomenon of complex formation between fibrinmonomer and fibrinogen degradation products was investigated by means of adsorption of FDP to insolubilized thrombin-modified fibrinogen (FM-ag). Since it could be demonstrated that there are different adsorption characteristics for early FDP and late FDP, the possibility of separation of FDP by means of affinity chromatography on FM-ag columns was evaluated using plasmic digests of 3H-Ac-labelled fibrinogen. The identification of FDP was performed by disc-electrophoresis. The results indicate that the adsorption of early FDP is comparable to the behaviour of fibrinogen, whereas late FDP show essential difference in the affinity towards FM-ag, evident by the result that fragment E adsorbs only to a minimal extents. Fragments D and E derived from fibrinogen as well as from non-crosslinked fibrin, revealed identical adsorption characteristics. Under specified conditions the procedure is suitable as a preparative method for the separation of fragments D and E.

Anticoagulant properties of extracellular slime substance produced by Staphylococcus epidermidis.

Slime produced by S. epidermidis strain KH 11 was extracted with phenol-saline. The saline phase was fractionated on a DEAE-Sepharose CL-6B column. The crude slime solution and its phenol-saline fraction were found to possess anticoagulant properties. They inhibited the coagulation of human plasma by thrombin, prolonged the activated partial thromboplastin time, but did not change the rate of plasma coagulation by reptilase. The anticoagulant effect of slime could be neutralized by rather high concentrations of protamine sulphate. In the presence of plasma, the staphylococcal slime also inhibited in a concentration dependent fashion the amidolytic activity of thrombin and factor Xa against synthetic chromogenic substrates. Both antithrombin III (AT III) and other plasma component(s), presumably heparin cofactor II, were required for the full expression of the slime effect. The anticoagulant action of slime was markedly less AT III dependent than that of heparin. The activity was resistant to heating (100 degrees C, 30 min). Slime and its fractions were stronger inhibitors of thrombin than of factor Xa. Fraction IV separated by DEAE-Sepharose chromatography and particularly rich in galactose and glucuronic acid had the highest inhibitory potency. It is conceivable that slime component(s) similar to glycosaminoglycans from other sources can carry the anticoagulant activity.

Studies on chemically modified fibrinogen.

Treatment of fibrinogen with maleic acid anhydride renders fibrinogen unclottable depending on the degree of modification of the molecule. According to radioactive studies the release of fibrinopeptides by thrombin or reptilase is undisturbed. The incoagulability is due to inhibition of the polymerization process of fibrinmonomers derived from modified fibronogen, mainly caused by the increase of electronegative charges upon the fibrogen molecule. According to discelectrophoretic analysis modified fibrinogen fails to produce fragments D and E following plasmic digestion, however, may be degraded to high molecular weight products. Modified fibrinogen reveals some similarities to abnormal fibrinogens in congenital dysfibrinogenemia with regard to its functional properties.

Standardized procedure for determination of staphylocoagulase.

A simple standardized procedure for the determination of staphylocoagulase, based on thrombin clotting time measurement with euglobulin, has been developed. A standard euglobulin solution containing 0.25% of clottable protein, 0.01 M epsilon-aminocaproic acid, and 20 units of heparin per ml is employed. One unit of staphylocoagulase is defined as the amount of the enzyme which clots this standard euglobulin solution in 25 sec.

Bovine fibrinogen modified with 3H-acetic anhydride (3H-AcOAc).

The method is described for acetylation of bovine fibrinogen with 3H acetic anhydride (3H-AcOAc). Preparations acetylated at pH 7.8 with 10 to 40 molar excess of 3H-AcOAc were found to contain 8 to 13 moles of acetyl residues per mole of fibrinogen. The content of clottable protein and ultraviolet (UV) spectra were unchanged as compared with control unlabeled preparations. The rate of clotting with thrombin was only slightly affected. The investigations on distribution of 3H-acetyl groups in products of proteolysis of acetylated fibrinogen by thrombin demonstrated a preferential acetylation of fibrinopetide A and absence of radioactive tracer in fibrinopeptide B. Incorporation of the label into fibrinopeptide A opens the possibility for application of fibrinogen labeled with 3H-AcOAc as a convenient substrate for selective investigation on the enzymatic phase of clotting.

INfluence of Pseudomonas aeruginosa proteases on prothrombin, plasminogen and fibrinogen.

Out of 136 strains of Pseudomonas aeruginosa tested, 101 produced a procoagulant protease and 70 a protease with fibrinolytic activity. Strain No. 1800 producing both these proteases served as a source of the crude enzyme mixture. The crude enzyme was isolated from the supernatant and subjected to fractionation by isoelectric focusing. Five active components were isolated. Activities of individual proteases were compared. It was found that for prothrombin activation a protease characterized by isoelectric point of 7.0 is responsible; this protease is different from both collagenase and elastase. Protease available at isoelectric point of 8.8 is plasminlike and is also not identical with collagenase and elastase.

Clotting and fibrinolytic activities of Bacteroides melaninogenicus.

106 strains of Bacteroides melaninogenicus ss. asaccharolyticus, intermedius and melaninogenicus were tested for production of extracellular blood clotting and fibrinolytic factors. 78.3% of tested strains caused clotting of rabbit plasma and 79.2% activated human plasminogen. Strains producing the clotting or fibrinolytic factor only were isolated. Both factors were quite active, as positive results for most strains were detected within one hour of incubation in controlled test system.

The nature of clotting and fibrinolytic activities of Bacteroids melaninogenicus.

From the supernatant of B. melaninogenicus ss. asaccharolyticus culture, a protein fraction was isolated by ethanol precipitation. The fraction was tested for the presence of clotting and fibrinolytic activities by application of quantitative techniques and specific substrates for measurement of prothrombin and plasminogen activation, and collagenase and elastase activity. It is postulated that ability of Bacteroides melaninogenicus ss. asaccharolyticus extracellular factors to clot fibrinogen and activate plasminogen, are due to a limited proteolysis by the proteolytic enzymes produced by this microorganism and not to the existence of specific B. melaninogenicus coagulase of plasminogen activator.