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A number of synthetic polymers and copolymers of the methacrylate type with a different content of sulfate, sulfamide, and carboxylic groups have been prepared. The anticoagulation activity of polymers thus obtained was compared with that of heparin. The highest anticoagulation activity was found with copolymer II containing 86 mol % of sulfate and 14 mol % of carboxylic groups. A surprisingly weak activity was observed with polymers containing sulfamide groups.

Genomic DNA from 90 Czechoslovak haemophilia A patients from 81 pedigrees was analysed by Southern blotting and hybridization with factor VIII cDNA probes. Three partial deletions of the factor VIII gene were identified and characterized: a 4.8 kilobase (kb) deletion eliminating exon 10 in one patient with severe haemophilia A without inhibitor, a 6.1 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in two related severe haemophiliacs, but only one of them produced inhibitor, and a 4.6 kb deletion eliminating the 3' part of intron 13 and the 5' part of exon 14 in a severe haemophiliac with high-titre inhibitor. Besides these three deletions, three different restriction site variants without apparent loss of DNA sequence were found.

von Willebrand disease (vWD) is the most common inherited bleeding disorder, which is highly heterogeneous ranging from asymptomatic laboratory abnormality to a life threatening bleeding. The condition is caused by a quantitative or qualitative deficiency of von Willebrand factor (vWF). Since 1994 it has been classified into six subgroups based on evaluation of the vWF level and function. Correct classification of vWD is required for its optimal management. vWD is inherited as a dominant or recessive trait linked to the vWF gene. However, the inheritance of type 1 vWD is not always linked to the vWF gene and novel modifying genes are expected to play a significant role in the type 1 pathophysiology. Laboratory tests required for correct type classification and for genetic analysis have long been awaited in Czech Republic. The purpose of this report is to inform about discriminatory and genetic tests recently made available at the Institute of Haematology and Blood transfusion.

Chronic liver disease is an important complication when treating hemophilia patients with factor VIII and IX concentrates. Hepatitis B (HBV) and C (HCV) virus infection is the major cause of chronic liver disease [5]. Chronic HCV infection is characterized by persistently or intermittently raised liver transaminases [4, 6].

Factor V Leiden (FVL) seems to be the most common hereditary thrombotic risk factor in the European population. Its prevalence in healthy Caucasian populations has been reported in a range between 3% and 7%. The aim of our study was to establish the prevalence of the defect in the Czech population.

Thrombosis is a common complication of many disorders and one of the most frequent death cause. There are six abnormalities accepted as genetic risk factors for venous thrombosis. Factor V Leiden mutation (FVL) is known as the most common of them. Its presence could increase the thrombotic risk five to ten times. That is the reason for FVL determination in young patients with thrombosis, especially idiopathic, in individuals with repeated thromboses or unusually sites. We also recommend family member’s examination in FVL carriers.

Hemophilia A is an X-linked bleeding disorder caused by a quantitative or qualitative defect of coagulation factor VIII. Since factor VIII has been cloned and several intragenic and linked DNA polymorphisms discovered, DNA analysis is an accepted and commonly used method for carrier testing in hemophilia A. Both a direct method using detection of mutation and an indirect method based on linkage between the disease and DNA polymorphism are used for this purpose. In this study, DNA samples of 110 hemophilia A patients from 99 families were screened for factor VIII gene mutation using Southern blot analysis; in seven families, mutations were detected. In 13 females from six families with identified mutation, the direct diagnosis of carriers was performed. Four intragenic (BclI, XbaI, BglI and MspI in F8C locus) and two linked polymorphisms (TaqI in DXS52 locus and BglII in DXS15 locus) were studied in members of 47 hemophilia A families. BclI-XbaI-BglI haplotypes were analyzed in 90 unrelated X chromosomes. Eighteen out of 31 females (58%) were heterozygous for at least one intragenic polymorphism, and 29 out of 31 females (94%) were heterozygous for at least one intra- or extragenic polymorphism tested. Carrier diagnosis was made in 15 out of 25 possible carriers (60%) based on intragenic and in an additional 3 out of 25 (12%) only on linked polymorphism.

Simple and efficient methods for the purification of functionally active clotting factors in good yields are still missing. The purpose of the present study was to design a chromatographic procedure for the isolation of protein C (PC) and its snake venom activators taking advantage of the high resolution, high speed of analysis and sensitive detection of high performance liquid chromatography.PC was purified from a human plasma concentrate containing vitamin Kdependent proteins using a Mono Q anion exchanger. Electrophoretic titration curve was used to serve as a guide for finding approximate conditions for separations. Elution profiles for vitamin K-dependent proteins were determined. PC was assayed by both immunological and functional methods. For the latter methods, protein C activators (PCA) were isolated from snake venoms of Agkistrodon c, -contortrix (ACC) and Agkistrodon c, mokasen.Both venoms (pooled samples)were found to contain at least two different PCA. AtpH 6.5> by simple one step procedures either an acidic PCA (on a Mono Q) or a basic PCA (on a Mono S) was isolated. A great diversity was found among venom samples from specimen originating from a small geographic area near Philadelphia (USA). In six out of the nine analysed ACC venoms only the acidic PCA was present.In conclusion, an optimum separationof protein C onthe Mono Q at pH 8.0 was proposed (usual recovery 90-95%).Optimalization of the salt gradient was considerably more effective than the pH changes. A great individual variability has to be taken into account when snake venoms are used as a source of protein C activators.

Two batches of lyophilized normal pooled HEPES-stabilized plasma according to ZUCKER [11] and LOELIGER [7] were prepared. The F VIII activity established by the activated partial thromboplastin test (APTT-VIII) was identical in both plasmas and amounted to 0.99 U/ml (95% confidence limits 0.87 to 1.17 U/ml). It did not change within 11 months in plasmas according to ZUCKER, and 6 months in those according to LOELIGER (further on not followed). We prepared four batches of lyophilized HERPES-stabilized substrate plasma (severe form of hemophilia A) according to ZUCKER. They were employed for 5 to 13 months in APTT-VIII along with the frozen 3.2% sodium citrate substrate plasmas. The results showed a satisfactory agreement. The lyophilized plasmas did not influence even the dose-response-line. On the ground of these results we consider the reported lyophilized plasmas to be suitable reagents in APTT-VIII technique.

The conversion of prothrombin by ecarin is independent of the presence of gamma carboxyglutamic residues on the N terminal of the molecule. Ecarin converts therefore also the acarboxylated precursor-PIVKA II. In a group of 347 patients under dicoumarol therapy of different intensity and duration PIVKA was detected in BaSO4 adsorbed PPP only in samples where prothrombin level (Quick's test) was lower than 50% of normal values. Increase in PIVKA did not correspond to the decrease of prothrombin. In some cases where the treatment was longer than two years no PIVKA was detected even when prothrombin was under 20%. This is explained by synthesis of partially carboxylated prothrombin molecules, which can be adsorbed. In liver diseases the ecarin test and Quick's time in native, untreated PPP showed about identical decrease of prothrombin level. This indicates that no PIVKA is released to plasma. The functional defect of the hepatocyte does not involve gamma carboxylation as long as vitamin K is provided. In patients with DIC ecarin test showed significantly higher level of thrombin activity than those obtained with Quick's test. It is known that during hypercoagulation stage of DIC prethrombin 1 and 2 are formed by the excess of thrombin. These split products of prothrombin are convertible by ecarin to meizothrombin 1 and 2. A positive ecarin test can be also due to acarboxylated precursors which are released during increased proteosynthesis triggered by consumption coagulopathy.