Simultaneous isolation of protein C activator, fibrin clot promoting enzyme (fibrozyme) and phospholipase A2 from the venom of the southern copperhead snake.

The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal-chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A2.

[Biological effects of interacting shock waves. A modeling study of the effects of interacting shock waves using erythrocyte hemolysis]. / Biologické úcinky interagujících rázových vln. Modelová studie úcinku IRV s vyuzitím stanovení hemolýzy erytrocytu.

AIM: The effects of high-energy shock wave on tissues were discussed in literature. The shock wave sources which were used for experiments were developed for stone fragmentation. The side effects of the applicators are generally low. Increase of shock wave pressure induce bigger negative pressure amplitude and this may cause significantly bigger side effects. We used tow shock waves, with time interval of 5 microseconds. In our opinion, the first shock wave causes acoustic inhomogenity in shock wave focus and therefore second shock wave acts in a region with different acoustic parameters. The second shock wave may loss energy in the focus area by dissipation or absorption. We termed the coupled shock waves as "interacting shock waves". METHODS AND RESULTS: Hemolysis of erythrocytes was used for examination of biological toxicity. Shock wave pressure was 80 MPa, the ratio of positive to negative pressure of single shock waves is 30. For correlation we applied 50 and 100 single shock waves and 50 or 100 couples of two shock waves. Hemolysis after 50 simple shock waves was 4.28 times lower compared with hemolysis after the same number of coupled shock waves (interacting shock waves). CONCLUSION: After 50 couples of shock waves hemolysis is 2.14 times higher compared with 100 single shock waves. This result suggests that the hypothesis of some interaction existing between two shocks applied in a very short time interval make future study rightful.

[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.

[Diagnosis of beta-thalassemia on the basis of HbA2 determination]. / Diagnostika beta-talasémie na základe stanovení HbA2.

The increased level of HbA2 is a reliable marker of heterozygous beta-thalassaemia. The levels of HbA2 measured by three different methods were compared and the ranges for the normal and for the heterozygous beta-thalassaemia were assessed. The levels of HbA2 2.76 +/- 0.47% for normal (30 blood donors) and 4.62 +/- 0.77% for beta-thalassaemia (50 patients) were obtained by the chromatographic method 2.61 +/- 0.42% HbA2 for normal (30 blood donors) and 5.82 +/- 0.89% HbA2 for beta-thalassaemia (46 patients) were assessed by electrophoresis on hydragel (Sebia) and 2.8 +/- 0.62% HbA2 for normal (30 blood donors) and 6.04 +/- 0.96% HbA2 (47 patients) were found when using cellulose acetate electrophoresis. An increased level of foetal Hb was found in nine patients with beta-thalassaemia. The diagnosis of beta-thalassaemia was confirmed by molecular genetic methods in all cases with an elevated HbA2 level, while a normal HbA2 level did not rule out heterozygous beta-thalassaemia.

[Structural variants in hemoglobin occurring in the Czech Republic]. / Strukturní varianty hemoglobinu nalezené v Ceské republice.

The authors present a review of clinical and laboratory findings of seven in the Czech Republic hitherto diagnosed structural haemoglobin variants. Unstable variants are found most frequently: Hb-Köln, Hb-St. Louis, Hb-Nottingham, Hb-E and Hb-Hradec Králové. The variant Hb-Hradec Králové (Hb-HK) or alpha 2 beta 2 115 (G17) Ala-Asp was newly detected. The great instability of Hb-HK chains makes classical diagnosis of Hb-pathy impossible. It was possible to identify it only at a molecular genetic level. A manifestation of Hb-HK instability is also the thalassaemic feature of the disease and the formation of Heinz bodies from free chains. The only representative of haemoglobins with a high oxygen affinity identified in this country was newly detected. It was given the name Hb-Olomouc or alpha 2 beta 2 86 (F2) Ala-Asp. This haemoglobin variant leads to erythrocytosis in father and son and the same clinical manifestations were recently described also in Japan. The last structural variant of haemoglobin found in this country is Hb-M Milwaukee or alpha 2 beta 2 67 (E11) Val-Glu which in our patients is manifested rather by haemolysis with formation of Heinz bodies than classical cyanosis. The cause of instability of Hb-M in our patients is not known. Hb-S was not diagnosed so far in the Czech Republic.

Hb Nottingham or alpha 2 beta 2 98 (FG5) Val-->Gly in a Czech child.

We report a fourth case of Hb Nottingham [alpha 2 beta 2 98 (FG5) Val-->Gly] observed in an 8-year-old girl in the Czech Republic with clinical and laboratory symptoms of severe hemolytic anemia. The unstable hemoglobin probably represents a de novo mutation, since the parents of the patient and the two siblings do not exhibit any hematological abnormalities. Splenectomy had a beneficial effect on the degree of hemolysis, as well as on the Hb level.

EDMA 2000 as a matrix for high-performance liquid chromatography of human haemoglobin chains.

An ethylenedimethacrylate polymer-based matrix (EDMA 2000) is described that shortens the time for separation of haemoglobin (Hb) chains to 30 min, compared with the use of C4 large-pore Vydac columns, commonly used for reversed-phase HPLC of Hb variants. EDMA 2000 was successfully used for the isolation of an abnormal beta-chain of the unstable Hb recently characterized as Hb Nottingham.

[Fibrinogen seen as an independent cardiovascular factor from the molecular aspect]. / Fibrinogen jako nezávislý kardiovaskulární faktor z molekulárního hlediska.

The blood protein fibrinogen is one of the main independent cardiovascular risk factors and it is very likely that an increased fibrinogen level is not the consequence of cardiovascular disease but its direct cause. Fibrinogen participates actively in many processes in the organism and undergoes various changes of the molecule but it is not known what is the molecular background, why even a slight increase of the fibrinogen level is so dangerous as regards increased risk of cardiovascular attacks. In the present work the authors compared, using monoclonal antibodies, the rate of release of fibrinopeptides A and B (FpA, FpB) by the action of thrombin from fibrinogen in solution and from fibrinogen adsorbed to a solid surface. The authors revealed that the rate of FpB breakdown from sorbed fibrinogen, contrary to fibrinogen in solution is comparable to the release rate of FpA and does not depend on the release of FpA (except for competitive inhibition). The release of FpB from sorbed fibrinogen thus takes place, contrary to fibrinogen in solution, at a significant recordable rate from the very onset of thrombin action. Fibrinogen adsorption to a solid surface is associated with conformation changes which cause this effect and which lead to the formation of a two-dimensional formation formed by fibrinogen after its interaction with the surface of activated platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

[New methods of isolating recombinant proteins]. / Nové zpusoby izolace rekombinatních bílkovin.

Recombinant proteins are isolated from very complex protein mixtures present in the producing cell. The isolation process involves in general four mutually interconnected stages: 1. release of the recombinant protein from the cellular environment, 2. preparation of the specimen for separation, 3. separation, 4. qualitative and quantitative analysis of the preparation. Each of these stages is formed by a complex series of methods which destroy the cellular wall, solubilize the specimen and involve the use of suitable precipitation, chromatographic, electrophoretic, immunochemical and other techniques for separation and analysis. In the submitted paper the authors describe a relatively simple isolation of recombinant peptide used for the preparation of a diagnostic kit for AIDS. From knowledge of the sequence of nucleotides in cDNA antisense peptides can be derived which have a high affinity with the isolated protein and they can be also used for affinity chromatography. An effective isolation technique is the use of mimetic ligands on the basis of textile dyes. By their combination and possible modification it is also possible to achieve separation of the required protein from contaminating substances. The mentioned highly specific methods can be combined with classical chromatographic techniques. During every step individual chromatographic fractions are tested by SDS electrophoresis in polyacrylamide gel incl. possible use of immunoblotting a specific staining.

A Czechoslovakian teenager with Hb E-beta zero-thalassemia [IVS-I-1 (G----A)] complicated by the presence of an alpha-globin gene triplication.

We have examined the molecular basis of three inherited hemoglobin (Hb) disorders present in a Czechoslovakian girl with a severe, transfusion-dependent, hemolytic anemia. She is heterozygous for Hb E (on a genetic background specific for Czechoslovakian families), heterozygous for the beta zero-thalassemia (thal) allele IVS-I-1 (G----A), and heterozygous for an alpha-globin gene triplication. The combination of these three undesirable traits results in a severe chain imbalance that is the basis of the serious hemolytic disorder observed in this teenager.

The action of a fibrin-promoting enzyme from the venom of Agkistrodon contortrix contortrix on rat fibrinogen and plasma.

The action of a fibrin-promoting enzyme isolated from the venom of A.c.contortrix was investigated. The ratio of fibrinopeptides A and B released was similar in isolated human and rat fibrinogen and human plasma, fibrinopeptide B always being released preferentially. However, no clotting occurred in rat plasma, and no fibrinopeptides were released even after prolonged incubation. The results suggest strong, fast-acting irreversible neutralization of the enzyme activity in rat plasma.

On the question of lowering the content of ferrihaemoglobin in infusable haemoglobin solutions.

The reducing effect of ascorbic acid and of borohydride upon ferrihaemoglobin present in native and chemically modified human and bovine stroma-free hemoglobins was investigated. Ferrihaemoglobin which had been freshly prepared from oxyhaemoglobin by treatment with potassium ferricyanate was fully reduced to ferrohaemoglobin. Full reduction of ferrihaemoglobin, however, could not be achieved with those haemoglobin samples which had a partially deteriorated conformation due to long time storage or chemical modification.