[Disorders of anticoagulation and fibrinolysis in monoclonal gammopathies--another mechanism of paraprotein interference with hemostasis]. / Poruchy antikoagulacních systému a fibrinolýzy u monoklonálních gamapatií--dalsí mechanismus interference paraproteinu s hemostázou.

We have investigated the possible interaction of paraprotein (pp) with anticoagulation mechanisms and fibrinolysis. Eighty four patients with monoclonal gammapathy (MG) were included to the study, 59 of them with multiple myeloma (MM). In 48.8% cases some defect was found. Decreased levels of antithrombin III (AT III) was observed in 13.3%, protein C (PC) in 18.3% and protein S (PS) in 13.5% of patients. Distribution between the free and the bound PS fraction remained normal. The most frequent abnormality found was the reduction of plasminogen (PLG) activity, which was observed in 35.1% and elevated levels of plasminogen activator inhibitor, detected in 42.3% of cases, respectively. Decreased plasminogen activator activity was observed in only one patient. The relationship between isotype and concentration of paraprotein and frequency of factor levels abnormalities was not found. The incidence of arterial and/or venous thrombosis was higher in patients with laboratory defect in comparison with the unaffected, however, the difference was not statistically significant. In contrast, the incidence of hemorrhagic complications was significantly lower in these patients (p < 0.01), although in most of them simultaneous defect of plasmatic coagulation and/or platelet functions was detected. We suggest, the interaction with both hemostatic and anticoagulation systems could result to "elimination" of inauspicious effect of pp on hemostasis. The impairment of anticoagulation systems and fibrinolysis is another type of paraprotein interference with hemostasis. It is also considered to be another pathogenetic mechanism of secondary deficiency of AT III, PC, PS and PLG.

[Functional determination of C1-inhibitor in plasma with a low level of prekallikrein]. / Funkcní stanovení C1-inhibitoru v plazmách se snízenou hladinou prekalikreinu.

The method of assessment of the C1-inhibitor (C1-INH) based on activation of plasma precallicrein (PC) by the fragment of Hageman's factor (HFf) was described in a previous publication (6). It was recommended as an alternative of already described methods used for assessment of C1-INH. To define the reliability of the recommended method in plasma with a reduced PC level the authors used plasma of mothers where during childbirth as a rule a steep drop of this proenzyme occurs. In a group of 23 parturient women the plasma PC levels were assessed and were within the range from 0.067-0.397 U/ml incl. only 6 plasma specimens where the PC level varied within the range of normal donors, i.e. 0.319-0.438 U/ml (mean value mean = 0.369 +/- 0.035). The mean value in plasma of the mothers was 0.237 +/- 0.100. In both groups the C1-INH was assessed by the recommended method and by the method of callicrein inactivation. In the group of mothers the results of C1-INH correlated with estimations in plasma where the PC level was within the range from 42.5 to 107.6% of the mean PC value of donors (r = 0.82). In plasmas of mothers whose levels varied from 18.2 to 50.2% of the mean value of PC donors the assessment of C1-INH by the method of HF fragment activation gave lower results in all instances. The dependence of C1-INH estimation on the amount of PC was demonstrated on deficient plasma with a defined PC level.(ABSTRACT TRUNCATED AT 250 WORDS)

[C1-inhibitor plasma levels in neonates and mothers during labor]. / Hladiny C1-inhibitoru v plazme novorozencu a matek pri porodu.

The authors investigated C1-inhibitor (C1-INH) plasma levels in blood collected from mothers during delivery: mothers with a normal pregnancy and a group of mothers who displayed during the perinatal period direct or indirect signs of infectious disease. Both groups, as compared with a group of healthy donors, had low C1-INH levels. The neonates were divided into two groups (normal children and children with perinatal risk of infection) with sub-groups of mature full-term neonates (38-41 weeks of gestation) and premature infants (29-36 weeks of gestation). In the group of normal infants the neonates had on average somewhat lower C1-INH levels, as compared with healthy donors, in premature neonates of this group the lowest mean C1-INH level was recorded. In this group the authors observed a correlation between the inhibitor level and the gestation period. The results of C1-INH assessment in the group of neonates with a perinatal risk of infection were different. In premature neonates a higher average C1-INH level was observed than in mature neonates and the relationship between the C1-INH level and the gestation period was a linear negative regression. The postnatal increase of C1-INH levels on the 1st to 5th day was more rapid in premature neonates of both groups.

[Functional determination of C1-inhibitors in human blood]. / Funkcní stanovení C1-inhibitoru v lidské krevní plazme.

The author describes the method of assessment of the C1-inhibitor, the principle being activation of plasma prekallikrein by Hageman factor fragment (HFf) to the active enzyme kallikrein which splits the specific chromogenic substrate NO-Pro-Phe-Arg-pNA. In the presence of acetone the influence of C1-INH is eliminated and the assessed amount of kallikrein corresponds to the prekallikrein plasma level. In a parallel estimation without acetone after formation of the C1-INH complex the residual amount of kallikrein is assessed. The difference between the two levels is proportional to the formed enzyme-inhibitor complex. The method was tested on plasma of healthy donors and the results were compared with assessment of C1-INH by a modification of Schapira's method (6) (correlation coefficient r = 0.88) and with Cullmann's method (8) (r = 0.61). The advantage of the proposed method is that it does not require commercially unavailable enzyme preparations, that it uses as activating agent HFf concentrate, prepared in the laboratory and that along with the C1-INH level also the plasma level of prekallikrein is assessed.

One-step preparation of Hageman factor fragment concentrate.

The Hageman factor fragment (HFf) concentrate was prepared from acetone and dextran sulphate activated human plasma by chromatography on a CM-Sephadex column. The preparation was free of the main kallikrein inhibitors--Cl-inactivator, alpha 2-macroglobulin, antithrombin III and alpha 1-antitrypsin. The HFf concentrate can serve as an activator of prekallikrein in patient plasma.

[Preparation of a prekallikrein concentrate and determination of its activator in blood derivatives]. / Príprava koncentrátu prekallikreinu a stanovení jeho aktivátoru v krevních derivátech.

The determination of an increased level of a fragment of the Hageman's factor (HFf) in preparations prepared from blood plasma can be an index of their potential reactivity in clinical application. The demonstration of the presence of HFf is based on the fact that the preparation containing this factor is able to activate the prekallikrein substrate. When the concentration of prekallikrein is sufficient, the amount of the developed active enzyme kallikrein is proportional to the amount of HFf. The amount of kallikrein is determined by cleaving the specific chromogenic substrate NO-Pro-Phe-Arg-pNA. The presence of salts negatively influences the rate of the activating stage of the reaction. The described preparation of the prekallikrein concentrate from the human blood plasma is a modification of the method of Levy and Sober) for the separation of the human immunoglobulin fraction. The blood plasma with an addition of hexadimethrinebromide is sorbed on DEAE Sephadex equilibrated with 0.0175 mol/l phosphate buffer pH 6.3 with hexadimethrinebromide in a vessel with an intact surface. Prekallikrein together with IgG are not sorbed on the anion exchanger under the above-mentioned conditions. The obtained supernatant can be employed as the prekallikrein substrate to determine the HFf activity in blood derivatives.

[Determination of prekallikrein in the plasma of neonates]. / Stanovení prekallikreinu v plazme novorozencu.

The authors assessed plasma prekallikrein (PK) after its conversion into the active enzyme kallikrein from the rate of breakdown of the specific chromogenic substrate NO-Pro-Pre-Arg-pNA. Activation of PK was achieved by the parallel contact method, using dextran sulphate (DS) and by direct activation by the Hageman's factor fragment (HFf). Contact activation assumes the presence of Hageman's factor (HF) and high molecular kininogen (HMW-K) in plasma. Both methods of activation and x = 0,451 +/- 0.146 U/ml in HFf activation). The mean PK value in neonates assessed by activation of DS was lower [mean = 0.105 +/- 0.068 U/ml] than the value assessed by activation of HFf [mean = 0.175 +/- 0.072 U/ml]. In adults the PK values varied at a significantly higher level, the results being higher when HFf was used as activator [mean = 0.346 +/- 0.087 U/ml in DS activation and mean = 0.451 +/- 0.146 U/ml in HFf activation]. The increase of mean values of PK assessed in umbilical blood and on the 3rd and 5th day after delivery was more marked in direct HFf activation. The correlation coefficient of both ways of assessment in neonates was r = 0.79, in the group of adults there was a close correlation r = 0.92. The lower correlation in neonates can be explained by the low level of co-factors, in particular HMW-K, necessary for complete activation of PK by the contact method. In some neonates the PK value assessed by DS expresses only the total activating plasma capacity and not the actual PK level.

Separation of the Hageman factor fragment from human serum albumin by chromatography on blue sepharose CL-6B.

The separation of the Hageman factor fragment (HFf) activity from human serum albumin by chromatography on Blue Sepharose CL-6B is described. The complete separation cannot be achieved in a single chromatography step due to complex formation between HFf and albumin.

The use of dipeptide-p-nitranilides for the study of aminopeptidase specificity.

The use of dipeptide-p-nitranilides for the study of 2 placental aminopeptidases separated on Sephadex G200 helped in establishing some regular features of their specifities. The high-molecular (320,000 daltons) one prefers Phe in position P'1 to Leu, whereas the lower-molecular aminopeptidase (145,000 daltons) prefers Leu. The high-molecular aminopeptidase splits very slowly the N-terminal Leu when Gly is in adjacent position. Leu-Gly-p-NA is therefore an inhibitor of this AP.

Rat liver homogenate hydrolases splitting N-acetyl-L-tyrosine ethyl ester.

Using gel filtration on Sephadex G 200, three fractions splitting N-acetyl-L-tyrosine ethyl ester (ATEE), termed I, S and II, were found in rat liver homogenate. In molecular size, electrophoretic mobility and thermolability, fractions I and II correspond to the ATEE-splitting enzymes contained in rat serum. The liver fraction S had a different surface electric charge and was relatively stable at high temperatures. Apart from ATEE, it splits certain ester and amide bonds of synthetic substrates. Purified fraction S increased vascular permeability in guinea pigs. Differential centrifugation of liver homogenate showed that fraction S and fractions I and II were localized differently in the subcellular particles.

Biological properties of rat serum hydrolases splitting N-acetyl-L-tyrosine ethyl ester.

An increase in the level of activity splitting N-acetyl-L-tyrosine ethyl ester (ATEE) was found in the plasma of rats with experimentally induced rheumatoid inflammation. The level of this activity rose paralled with development of the inflammation. Homogenate of inflamed rat paws was found to contain a raised content of the high molecular weight fraction. Which was found to contain a raised content of the fraction I (splitting ATEE) causes an increase in vascular permeability and releases active kinins from plasma kininogens. These properties were also found, on a smaller scale, in serum fraction II. The results show no parallel between ATEE-splitting activity and the magnitude of the biological response.