Petr Dulíček

Thrombosis in thrombocythemic Ph-myeloproliferations is associated with higher platelet count prior to the event: results of analyses of prothrombotic risk factors from a registry of patients treated with anagrelide

Controversies still exist regarding definition of the thrombotic risks in Ph‐ (BCR/ABL1‐) myeloproliferative disorders with thrombocythemia (MPD‐T). Platelet counts at diagnosis are currently not taken as a risk factor of thrombosis. In our cohort of 1179 patients with MPD‐T, prospectively registered for anagrelide treatment, we found that the median platelet count prior to the thrombotic event was significantly higher than at time points without any ensuing thrombosis (453 vs. 400 × 109/L, P < 0.001), albeit higher platelet counts at diagnosis tended to be connected with fewer thrombotic events (in contrast to WBC counts at diagnosis). The JAK2V617F mutation predicted both arterial and venous events, while age >65 yr, hypertension, diabetes mellitus, smoking, elevated triglyceride and homocysteine levels predicted arterial events only. For venous events, the specific thrombophilic risk factors (factor V ‘Leiden’ and others), antiphospholipid antibodies, and elevated factor VIII levels played a major role. During anagrelide treatment (± aspirin), we documented a decrease in both venous (6.7‐fold) and arterial events (1.8‐fold), while bleeding (mostly minor events) increased twofold compared to history. Our results suggest that keeping platelet counts at low levels may be a meaningful therapeutic measure to prevent thrombosis, although their counts at diagnosis lack any prognostic value.

Venous Thromboembolism in Young Female While on Oral Contraceptives: High Frequency of Inherited Thrombophilia and Analysis of Thrombotic Events in 400 Czech Women:

Oral contraceptive use is a common risk factor for venous thromboembolism in women of reproductive age. The presence of inherited thrombophilia further increases this risk. Methods: We analyzed a large group of 400 Czech women with venous thromboembolism in association with oral contraceptive with regard to duration of use at the time of manifestation of venous thromboembolism, the frequency of inherited and acquired thrombophilia, the frequency of eliciting risk factor for thrombosis including immobilization, surgery, administration of plaster cast, long travel, and so on, and the type of thrombosis. The mean age of the women was 26 years, and the average duration of use was 45 months at the onset of thrombosis. Results: Venous thrombosis solely due to the pill occurred in 57% of the women, and in the other 43%, an additional transient eliciting factor was recognized. Among the clinical manifestation, distal thrombosis prevailed (N = 231, 58%) followed by proximal deep vein thrombosis (N = 65, 16%), pulmonary embolism (N = 21, 5%), and thrombosis in unusual sites (N = 20, 5%). Inherited or acquired thrombophilia was diagnosed in 195 (49%) women: factor V Leiden mutation in 35%, congenital deficiency of antithrombin in 1.8%, protein C in 0.8%, protein S in 1%, F IIG20210A in 5%, and antiphospholipid syndrome (APS) in 5.3%. Among the most common risk factors were immobilization of lower limb, minor and major surgery, and trauma. Conclusion: The results confirm that venous thromboembolism is a multifactorial disease in which thrombophilia screening is needed in young symptomatic women on the pill with thrombosis. The results also emphasize the value of proper thromboprophylaxis in women while on oral contraceptive in situations of increased risk for venous thromboembolism.

Risk of Thrombosis in Patients Homozygous and Heterozygous for Factor V Leiden in the East Bohemian Region

A retrospective investigation on the frequency of thrombotic events in 227 heterozygous and 16 homozygous carriers for the factor V R506Q mutation (factor V Leiden) from 102 unrelated families in the East Bohemian region is reported. A majority of 130 of the 227 (57%) heterozygous and a minority of 5 of 16 (31 %) homozygous carriers for the factor V R506Q mutation remained asymptomatic. Deep venous thrombosis without pulmonary embolism in a minority was the main and the most frequent clinical manifestation in 97 of 243 carriers for the factor V R506Q mutation. Deep venous throm bosis occurred in 92 of 227 (41%) heterozygous and in 11 of 16 (69%) homozygous carriers of the factor V R506Q. Spontane ous thrombosis prevailed in symptomatic men. Thrombosis in women usually occurs during risk situations well known to elicit venous thrombosis. Oral contraceptives and pregnancy were significantly the most frequent risk factors in female car riers of the factor V R506Q mutation, which in fact can readily explain the lower average age for the first thrombotic event of 30.6 years in women compared to 37.1 years in men.

Analysis of Risk Factors of Stroke and Venous Thromboembolism in Females With Oral Contraceptives Use

Thrombotic diathesis has been a well-known complication of oral contraceptive use for more than 50 years. This is true not only for venous thrombosis but also for an arterial one. The etiology is usually multifactorial and depends on several additional risk factors. We analyzed the prevalence of inherited and acquired thrombophilia in a cohort of 770 females who had a thrombotic event in association with oral contraceptive use (700 women with venous thromboembolism [VTE], 70 with stroke). Moreover, we tried to identify additional risk factors. Inherited thrombophilia was found in 44.5% with higher frequency in the cohort with VTE (42%) than in females with stroke (24%). The most frequent finding was factor V Leiden. Cigarette smoking was significantly more frequent in the group with stroke (50% vs 25%). The prevalence of cigarette smoking in the group with VTE did not exceed the frequency in general population. Women on oral contraceptive pills have higher risk of venous as well as arterial thrombosis. The risk of venous thrombosis is increased in females with inherited thrombophilia, whereas those with some additional acquired risk factors (especially smoking) may be predisposed to arterial thrombosis. However, the absolute risk of thrombosis in healthy women is low, far less than the risk of unintended pregnancy. Moreover, the risk may be reduced by keeping some rules before the prescription of the pills, healthy life style, and a proper choice of contraception.

The Purple Toe Syndrome in Female With Factor V Leiden Mutation Successfully Treated With Enoxaparin

Purple toe syndrome is a rare complication of warfarin therapy. It occurs usually after 3 to 8 weeks of therapy and it is caused by cholesterol emboli from atheromatous plaque. Sudden onset of pain in affected area, typically in toes and feet, is the main characteristic of the syndrome. We describe a case of a 65-year-old female with purple toe syndrome after 6 weeks of warfarin. Indication of warfarin was a proximal deep venous thrombosis, which developed after prolonged immobilization. Factor V (FV) Leiden and persistent high FVIII activity were found as additional eliciting factors for venous thromboembolism. After warfarin withdrawal and enoxaparin treatment, symptoms disappeared promptly but a slight discoloration of the toe persists.

Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations

AIMS The aim of our study is to present a novel approach for preparing a compound heterozygous reference material (hetRM) using gene synthesis technology with inverted insertion of wild-type and mutant fragments into a single cloning vector. Factor II (G20210A) and Factor V (G1691A Leiden) gene mutations were used as an experimental model. METHODS During the gene synthesis, DNA fragments were aligned in the following order: G1691 FV wild-type forward strain, G20210 FII wild-type forward strain, 1691A FV mutant reverse strain, 20210A FII mutant reverse strain. The complete chain was inserted into a pIDT SMART cloning vector and amplified in an E. coli competent strain. For assessing hetRM characteristics and commutability, we used real-time PCR with subsequent melting curve analysis, real-time PCR with hydrolysis probes, allele-specific amplification, reverse hybridization, and dideoxynucleotide DNA sequencing. RESULT All five methods yielded concordant results of DNA analysis of the hetRM. Differences in real-time PCR cycle threshold values after six-months of storage at -80 °C were not statistically significant from those obtained from freshly prepared hetRM aliquots, which is a good indication of their stability. CONCLUSION By applying the procedures of gene synthesis and cloning technology, we prepared and verified a model genetic reference material for FII G20210A and FV G1691A testing with a compound heterozygous genotype. The hetRM was stable, commutable, and available in large quantities and in a wide concentration range.

CASE 1-2013: FATAL MANIFESTATION OF MYELOPROLIFERATIVE DISEASE

Myeloproliferative disorders (MPDs) are defined as clonal stem cell diseases. the cardiovascular system is involved in 4 to 21% of MPD cases, presenting as acute ischemic coronary artery disease, valvular heart involvement, pericardial involvement, aortitis, thrombosis of major vessels, pulmonary embolism, systemic and pulmonary hypertension. Acute coronary syndrome may be the first manifestation of myeloproliferation.