Predrag Miljic

A novel prothrombin mutation in two families with prominent thrombophilia--the first cases of antithrombin resistance in a Caucasian population.

V. DJORDJEV IC ,* M. KOVAC,†‡ P . MIL J IC ,†§ M. MURATA, ¶ A. TAKAGI , ¶ I . PRUNER ,* D . FRANCUSK I , * T . KOJ IMA ¶ and D . RADOJKOVIC* *Institute of Molecular Genetics and Genetic Engineering, University of Belgrade; †Faculty of Medicine, University of Belgrade; ‡Hemostasis Department, Blood Transfusion Institute of Serbia; §Clinic of Hematology, University Clinical Center, Belgrade, Serbia; and ¶Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan

Increased risk of venous thromboembolism in patients with primary mediastinal large B-cell lymphoma

BACKGROUND Primary mediastinal large B-cell lymphoma (PMBCL) is a rare subtype of diffuse large B-cell lymphoma, arising in the mediastinum. Compression of large mediastinal vessels is common in patients with PMBCL, predisposing these patients to venous thrombosis. The incidence of this complication is still unknown. MATERIALS AND METHODS We conducted a retrospective chart review of 42 consecutive patients diagnosed with PMBCL at a Clinic for hematology, Clinical Center of Serbia between 1999 and 2009 to identify the frequency and risk factors for venous thromboembolic disease (VTE) and its correlation with survival. RESULTS In the subgroup of patients with thrombosis (VTE subgroup) were 15/42 patients (35.7%): 14 patients had deep venous thrombosis and 1 had pulmonary embolism. Ten patients had a thrombosis at the moment of diagnosis PMBCL, while in five thrombosis occurred during the course of the disease. According to hemostasis variables, patients in the VTE subgroup had significantly higher fibrinogen (P=0.02) and D-Dimer (P<0.001). Also, patients in the VTE subgroup had significantly larger diameter of mediastinal tumor mass (P=0.01) and the incidence of syndrome venae cava superior (P=0.009). The median survival rate in the VTE subgroup was 45 months (95% CI; 22 to 68 months) while in the NVTE it was 93 (95% CI; 46 to 140 months), (log rank P=0.058). CONCLUSIONS VTE is a common complication in PMBCL patients and according to our results negatively influences survival. Use of antithrombotic prophylaxis may be considered together with chemotherapy, especially in those with bulky mediastinal tumor mass.

Procarboxypeptidase U (TAFI) contributes to the risk of thrombosis in patients with hereditary thrombophilia

INTRODUCTION It is considered that high plasma levels of procarboxypeptidase U (proCPU or TAFI) can promote the development of thrombosis, but data comparing proCPU levels in thrombophilia carriers and healthy subjects are rather scarce. Moreover, the results of previous studies on the risk of thrombosis related to high proCPU concentration in this population were not consistent. Although the 325 polymorphism of proCPU has a significant effect on the CPU half-life, its influence on the risk of thrombosis or spontaneous pregnancy loss in carriers of hereditary thrombophilia is not clear. MATERIALS AND METHODS The study population consisted of 144 thrombophilic patients (94 heterozygous and 10 homozygous carriers of FV Leiden, 26 heterozygous carriers of the prothrombin G20210A variation and 14 double carriers of FV Leiden and FII variation) and 69 healthy controls. RESULTS The results show that patients with inherited thrombophilia have a tendency toward lower mean proCPU plasma levels compared to healthy controls, however, this difference was only significant in carriers of FII G20210A (p=0.014). A higher frequency of the most stable Ile325Ile proCPU was seen among carriers of FII G20210A mutation compared to the control group (19% vs 7%; p=0.186). In the second part of the study proCPU as a risk factor for thrombosis was evaluated. In heterozygous carriers of FV Leiden or FII G20210A high levels of proCPU conferred to an almost 4-fold increased risk for spontaneous onset thrombosis. The more stable Ile325Ile proCPU seems to impose a higher risk for clinical manifestation of the thrombophilic condition. Finally, a significant positive correlation between F1+2 and proCPU concentration was seen. CONCLUSION The increased risk of thrombosis in thrombophilia patients is not only ascribable to an increased thrombin generation, but also high levels of proCPU and the presence of the 325Ile genotype tip the balance towards thrombotic tendency even further.

Factor V Leiden, FII G20210A, MTHFR C677T mutations as risk factors for venous thrombosis during pregnancy and puerperium

BACKGROUND Venous thrombosis is the most common cause of obstetric morbidity and mortality during pregnancy and puerperium. The incidence of pregnancy-associated venous thrombosis varies from 1 in 1000 to 1 in 2000 deliveries. Factor V G1691A (FV Leiden), FII G20210A and MTHFR C677T mutations are the most common genetic risk factors for thromboembolism. The aim of this study was to establish the presence of these risk factors in a group of women with an episode of deep venous thrombosis during pregnancy or puerperium. METHODS The study was carried in a group of 45 women with the first episode of deep venous thrombosis during pregnancy or puerperium. The patients with antiphospholipid antibodies, antithrombin III, protein C or protein S deficiency, and autoimmune and malignant diseases were excluded from the study. FV Leiden, FII G20210A, and MTHFR C677T mutations were detected by polymerase chain reaction, followed by digestion with specific restriction enzymes. RESULTS Twenty heterozygous carriers of the FV Leiden mutation and one homozygous carrier were detected, which represents the frequencies of 44.4% and 2.2%, respectively. For the FII G20210A mutation, six heterozygous carriers were identified, giving the frequency of 13.3%. The MTHFR C677T mutation was observed in 31 patients (22 heterozygous and 9 homozygous carriers) which represents the frequencies of 48.9% and 20%, respectively. CONCLUSION Our study suggested that the obligatory testing for FV Leiden and FII G20210A mutations was strongly recommended in women with history of venous thrombosis during pregnancy and puerperium. We found a slight effect of MTHFR 677T allele, but it should be considered in association with other risk factors.

Thrombotic events in acute promyelocytic leukemia

INTRODUCTION Thrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to 10-15%. MATERIALS AND METHODS We retrospectively analyzed the data on TE appearance in 63 APL patients. RESULTS TE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P=0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P=0.046), PT (P=0.022), aPTT (P=0.044), ISTH DIC score (P=0.001), bcr3 (P=0.02) and FLT3-ITD (P=0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3-ITD mutation (P=0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P=0.05). Regarding risk factors for arterial TE we failed to identify any. CONCLUSIONS We have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score<5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G.

Intracardiac thrombosis and fever possibly triggered by ovulation induction in a patient with antiphospholipid antibodies

We report on a 28-year old patient with polycystic ovary syndrome (PCOS) who presented with fever and laboratory markers of inflammation. Her medical history was relevant for multiple ovulation inductions (OI) and ovarian hyperstimulation syndrome (OHSS). She had two miscarriages and one preterm delivery. Intracardiac thrombosis was diagnosed in the presence of antiphospholipid antibodies. We suggest that primary antiphospholipid syndrome (APS) was possibly triggered by OI.

Haemostatic abnormalities in treatment-naïve patients with Type 1 Gaucher's disease

There is a paucity of data on the effects of enzyme replacement therapy (ERT) on the coagulation abnormalities and platelet function of patients with Gauchers disease (GDPs) and much of this data are controversial. This study investigates the haemostatic parameters in treatment-naïve GDPs and the effects of ERT. 31 Serbian treatment-naïve type 1 GDPs (M/F 17/14; median age 49 years, splenectomized 9/31) were studied. The complete blood count, prothrombin time (PT), activated partial tromboplastin time (aPTT) and coagulation factors were measured using the standard methods. Platelet aggregation was assessed with a whole-blood aggregometer. Splenic volumes were assessed using computer tomography. Twenty-one patients were treated with ERT (Imiglucerase). The haemostatic parameters were assessed after 6, 12 and 24 months (ERT6, 12, 24). Initially bleeding episodes were registered in 10/31 GDPs. Median platelet count was 108 × 109/L; 22/31 GDPs were thrombocytopenic. The PT and aPTT values were abnormal in 16/31 and 13/31 GDPs, respectively. Platelet aggregation abnormalities were recorded in 19/31GDPs. Median platelet aggregation was reduced in response to adenosine-diphosphate 5 µmol/L (ADP5 0.46) and collagen 5 µmol/L (Col5 0.47). Splenic volume inversely correlated with the platelet count and a reduced response to arachidonic acid (AA), Col5 and ADP5 (p < 0.05). The splenectomized GDPs had a significantly lower platelet aggregation to Col10 (p < 0.05). Bleeding GDPs had a significantly lower platelet count, higher chitotriosidase levels and a greater splenic volume compared to non-bleeding patients (p < 0.01). ERT: The number of bleeding GDPs had significantly decreased by ERT6 (1/10; p < 0.01). The platelet count had significantly increased by ERT6 (ERT6 180 × 109/L, p < 0.01). The PT increased significantly from ERT0 to ERT24 (PT0 65%, PT24 81%; p < 0.05). The von Willebrand factor had increased significantly by ERT6 and ERT24 (ERT0 56%, ERT6 70%, ERT12 70%, ERT24 86%; p < 0.01). The number of GDPs with abnormal platelet aggregation had decreased significantly by ERT6 (10/19; p < 0.05). Platelet aggregation on ADP10 and AA significantly increased by ERT6 (ADP10: ERT0 0.75, ERT6 0.8 p < 0.01; AA: ERT0 0.7, ERT6 0.8 p < 0.05). In conclusion, platelet dysfunction and coagulation abnormalities were found in a considerable number of our GDPs. The absence of severe bleeding episodes suggests that the haemostatic system is sufficiently balanced and therefore the exact mechanism of the etiology of these abnormalities need to be fully clarified. ERT resulted in the cessation of bleeding and marked increase in platelet count, PT, vWF and platelet aggregation.

Growth hormone replacement normalizes impaired fibrinolysis: New insights into endothelial dysfunction in patients with hypopituitarism and growth hormone deficiency

BACKGROUND Cardiovascular morbidity in adult patients with growth hormone deficiency (GHD) and hypopituitarism is increased. Clustering of cardiovascular risk factors leading to endothelial dysfunction and impaired fibrinolysis has also been reported and may account for progression to overt vascular changes in these patients. However, effect of long lasting GH replacement therapy on fibrinolytic capacity in GH deficient patients has not been investigated so far. OBJECTIVE To investigate fibrinolysis before and after challenge with venous occlusion in GHD patients with hypopituitarism before and during one year of growth hormone replacement. DESIGN Hospital based, interventional, prospective study. INVESTIGATED SUBJECTS Twenty one patient with GHD and fourteen healthy control subjects matched for age, sex and body mass index (BMI). METHODS Anthropometric, metabolic and fibrinolytic parameters were measured at the start and after three, six and twelve months of treatment with human recombinant GH. RESULTS At baseline GHD patients had significantly impaired fibrinolysis compared to healthy persons. During treatment with GH, significant changes were observed in insulin like growth factor 1(IGF-1) [from baseline 6.9(2.4-13.5) to 22.0(9.0-33.0) nmol/l after one month of treatment; p<0.01] and fibrinolysis. Improvement in fibrinolysis was mostly attributed to improvement of stimulated endothelial tissue plasminogen activator (t-PA) release in response to venous occlusion [from baseline 1.1(0.4-2.6) to 1.9(0.5-8.8) after one year of treatment; p<0.01]. CONCLUSION Growth hormone replacement therapy has favorable effects on t-PA release from endothelium and net fibrinolytic capacity in GHD adults, which may contribute to decrease their risk of vascular complications.

+1040 C/T polymorphism in coding region of thrombin-activatable fibrinolysis inhibitor gene and the risk of idiopathic recurrent fetal loss.

Recurrent fetal loss (RFL) is common health problem affecting up to 5% of women of reproductive age. It has been shown that plasma thrombin-activatable fibrinolysis inhibitor (TAFI) concentrations increase during pregnancy and return to baseline levels soon after delivery. The +1040C/T single nucleotide polymorphism in coding region of TAFI gene is associated with TAFI blood levels. The aim of our study was to investigate the relationship between +1040C/T polymorphism in TAFI gene and idiopathic RFL. Study was carried out in a group of 120 women (61 controls and 59 women with idiopathic RFL). The +1040C/T polymorphism was detected by restriction fragment length polymorphism PCR. Increased frequency of +1040T/T genotype was observed in a study group, but without statistically significant difference. Carriers of T/T genotype have increased risk of fetal loss by 1.23-fold, compared with carriers of C/C (95% CI 0.462–3.277; P = 0.7) and 1.34-fold compared with carriers of C/T genotype (95% CI 0.501–3.601; P = 0.6). C allele is associated with reduced risk of recurrent fetal loss compared with T allele (OR 0.91; 95% CI 0.545–1.533; P = 0.7). In conclusion, we observed increased frequency of +1040T/T genotype in a patient group, suggesting that this genotype could be potential risk factor for idiopathic RFL. Further investigation should be carried out in order to establish the role of this polymorphism in the etiology of idiopathic recurrent miscarriages.

Clinical and biochemical characterization of the prothrombin Belgrade mutation in a large Serbian pedigree: new insights into the antithrombin resistance mechanism

Essentials Prothrombin Belgrade mutation leads to antithrombin resistance. Clinical and biochemical phenotypes in a large family with this mutation were investigated. In carriers, we detected decreased factor II activity and increased endogenous thrombin potential. Prothrombin Belgrade mutation represents a strong prothrombotic risk factor.