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. SUMMARY: Background The recently reported c.1787G>A mutation in the prothrombin gene leads to Arg596Gln replacement in the protein molecule (prothrombin Belgrade). This substitution impairs binding of antithrombin to thrombin and results in inherited thrombophilia, known as antithrombin resistance. Objectives We aimed to elucidate the clinical and biochemical characteristics of thrombophilia associated with antithrombin resistance in a large Serbian family with the prothrombin Belgrade mutation. Patients and methods Nineteen family members were investigated, among whom 10 were carriers of the c.1787G>A mutation. In all subjects the clinical phenotype was determined and laboratory investigations of hemostatic parameters were performed. Results Six out of the 10 mutation carriers developed thromboembolic events, mainly deep venous and mesenteric vein thrombosis. The median age of the first thrombotic event was 26.5 (12-41) years, whereas the incidence rate of first thrombosis was 2.2% per year. In all mutation carriers prothrombin activity was significantly decreased in comparison with non-carriers, clearly distinguishing each group. However, the presence of the mutation did not affect the prothrombin antigen level in plasma. The endogenous thrombin potential was significantly increased in all carriers in comparison with non-carriers, indicating the presence of blood hypercoagulability. Interestingly, levels of D-dimer and the F1+2 fragment were similar in both groups. Conclusions Although rare, the prothrombin Belgrade mutation represents strong thrombophilia with early onset of thrombosis in the investigated family. According to our results, decreased prothrombin activity may be a simple screening test for detection of this mutation in thrombotic patients.

Proliferative characteristics of Philadelphia-negative myeloproliferative neoplasms - clinical implications.

INTRODUCTION: Philadelphia-negative myeloproliferative neoplasms (Ph- MPN) are characterized by overproduction of one or more blood cell lines. METHODS: We studied the proliferative characteristics of 91 patients with de novo Ph- MPN. Colony-forming cells (CFC) and endogenous colonies (EC), from bone marrow (BM) and/or peripheral blood (PB), were analyzed by colony assay based on methylcellulose. The level of circulating CD34+ cells was determined by flow cytometry. RESULTS: The total number of PB CFC in primary myelofibrosis (PMF) was increased compared to the control sample (P < 0.01) and essential thrombocythemia (ET) (P < 0.05). The highest number of BM and PB EC was observed in polycythemia vera (PV) (P < 0.01). Increased levels of CD34+ cells characterized early-prefibrotic (57%) and advanced-fibrotic PMF (90%) as compared to PV (34%) and ET (32%) (P < 0.01). In the whole Ph- MPN group, the total number of PB CFC (P < 0.01), PB EC (P < 0.05), and CD34+ cells (P < 0.01) correlated with the degree of BM fibrosis. Higher levels of circulating CD34+ cells in PMF correlated with the total number of PB EC (P < 0.05) and degree of BM fibrosis (P < 0.01). CONCLUSIONS: Exploration of the PB proliferative characteristics of Ph- MPN on diagnosis may be helpful in revealing early-prefibrotic PMF. Monitoring the levels of circulating CD34+ cells may provide a sensitive indicator of fibrotic evolution in PV and PMF.

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.

Multiorgan thrombotic disorder in a young patient with primary antiphospholipid syndrome (APS) and ovarian tumor.

Catastrophic antiphospholipid syndrome (CAPS) is a life-threatening condition with high mortality rate besides aggressive multimodal treatment. Underlying triggers of "thrombotic and cytokine storm" include pregnancy, inflammation, trauma, surgery, and infection. The authors present a case of a young female patient with primary antiphospholipid syndrome (APS) who was admitted to the hospital due to abdominal pain caused by ovarian tumor with elevated tumor markers. After the prophylactic anticoagulants and antibiotic treatment, surgery was performed. Suddenly after treatment, her clinical status deteriorated and she died regardless of intensive immunosupresive and anticoagulant therapy attempts. This condition requires all clinical awareness, timely diagnosis, and therapeutical approach, including a better understanding of the pathophysiology that leads to CAPS.

Antiribosomal-P protein antibodies in a patient with systemic lupus erythematosus and non-Hodgkin's lymphoma: more than coincidental finding?

Patients with systemic lupus erythematosus (SLE) are at an increased risk of lymphomas, but mechanisms underlying this association are obscure. Recently, it has been shown that antiribosomal-P protein (anti-P) antibodies cross-react with phospholipids and enhance the production of cytokines which may influence lymphomagenesis. We report a 46-year-old woman who suffered high grade diffuse large B-cell non-Hodgkin's lymphoma (DLBCL) 28 months after the diagnosis of SLE. Development of lymphoma was associated with occurrence of serum monoclonal IgM, and pronounced prolongation of phospholipid-dependent clotting tests. Anti-P IgG antibodies were highly positive both on HEp-2 cells and in ELISA test. Anticardiolipin, anti-beta2 glycoprotein I, and antiprothrombin IgM antibodies have also been found in high concentrations. Complete remission of DLBCL and SLE, with normalisation of clotting tests, and disappearance of M component was achieved with administration of rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone. The progression of SLE to DLBCL associated with presence of anti-P antibodies has not been previously reported. This association may not be coincidental, but further investigations are required to confirm this hypothesis.

Serum lipids and anti-oxidized LDL antibodies in primary antiphospholipid syndrome.

OBJECTIVE: The link between specific antibodies and atherogenesis in primary antiphospholipid syndrome (PAPS) is less strong than for thrombosis, although clearly the two processes are related and thrombosis is the main complication of atherosclerosis, a process known as atherothrombosis. The aim of this study was to investigate the influence of serum lipid levels and anti-oxidized LDL (oxLDL) antibodies on the clinical features of 42 patients with PAPS (mean age 40.45+/-13.37; 32 women and 10 men), and to compare them with 47 control subjects (mean age 39.68+/-13.93; 33 women and 14 men). METHODS: Total cholesterol, HDL and triglyceride concentrations were determined by enzymatic methods. LDL was calculated according to the Friedwald formula. Anticardiolipin, anti-oxidized LDL and anti-Beta2glycoprotein I antibodies were detected by ELISA. RESULTS: A significant association was found between arterial events and triglyceride, LDL and cholesterol concentrations, but multivariate analysis showed that cholesterol concentrations were the most important predictor of arterial events (p=0.012). Cerebrovascular insults were the most significantly associated with cholesterol concentrations (p=0.011). Myocardial infarctions were more frequently present in patients more than 40 years of age (p=0.032). No significant association of the investigated parameters with venous thromboses was found. Recurrent abortions were not associated with the presence or concentrations of the investigated parameters. Although patients had increased concentrations of anti-oxLDL antibodies, no significant association was found between the titres of anti-oxLDL antibodies and clinical features of APS. CONCLUSION: In patients with PAPS, lipid concentrations are a better predictor for arterial events than anti-oxLDL antibodies.

[Myeloproliferative diseases as causative agents of portal and hepatic veins thrombosis].

Thrombosis of portal and hepatic veins is one of the most severe complications and most important causes of death of patients with chronic myeloproliferative diseases. Based on results of the past studies, myeloproliferative diseases were the causes of hepatic veins thrombosis in 30% and portal vein thrombosis in 20% of patients. The study presented 4 patients with myeloproliferative diseases complicated by thrombosis of splanchnic veins, aiming at the illustration of issue complexity in diagnostics and therapy. Two patients with portal vein thrombosis and recurring hemorrhage from esophageal varicosity were described. The first case was planned for shunting, while another case sustained bleeding on what account his anticoagulant therapy was discontinued, but it caused mesenterial thrombosis resulting in lethal outcome. Another two patients had hepatic veins thrombosis. Due to frequent, life-threatening bleeding from the esophageal and gastric varices, a patient with chronic Budd-Chiari syndrome and lineal vein thrombosis underwent mesocaval shunting. An immediate postoperative period was manifested by multiple thrombosis and hemorrhages that ended in his death. A patient with the acute Budd-Chiari syndrome was administered myelosuppressants and anticoagulants on time so reperfusion was restored. In myeloproliferative diseases, thrombosis of portal and hepatic veins gives rise to excessive portal hypertension with profuse hemorrhage from the esophageal and gastric varicosity which is difficult to manage because of complex coagulation disorders.

New approach of the treatment of von Willebrand's disease during pregnancy.

AIM: The aim of the study was to analyze the effectiveness of the application of DDAVP (desmopressin) and Hemate P with cryoprecipitate pre- and postpartum in patients with von Willebrand disease. METHODS: We monitored 32 patients with von Willebrand disease during the study period 1993-2003. DDAVP was applied in the 36th/37th week of gestation and cryoprecipitate and fresh frozen plasma were applied 1 day before and 3 days after delivery. DDAVP treatment continued for 4 weeks. Factor VIII (Hemate P) at the day of delivery RESULTS: No complications occurred in the studied population. CONCLUSION: Precipitation of DDAVP, Hemate P, and cryoprecipitate may help in the treatment of pregnant women with von Willebrand disease.

[Retropancreatic hematoma mimicking a tumor in a female patient with liver amyloidosis]. / Retropankreasni hematom koji je imitirao tumor kod bolesnice s amiloidozom jetre.

Bleeding in patients with amyloidosis is considered as a frequent complication appearing in up to 40% of cases. It may be spontaneous or provoked. Spontaneous bleeding in the skin or in the gastrointestinal tract is the most frequent. Spontaneous bleeding in the retroperitoneum is rare. It may be diffuse, with possible lethal outcome, or localised, causing haematoma, as in our patient. We present a 62-year old woman with a three-month history of spontaneous, subcutaneous bleeding. No laboratory or other findings could explain the cause of bleeding, as the haemostatic tests were within or close to normal limits. US and CT performed for right subcostal pain discovered a tumour attached to the back of the head of pancreas. Due to mild coaugated hyperbilirubinaemia the "tumour" was held to be the cause of patient's difficulties. However, at operation it turned out to be a retropancreatic haematoma, which was removed and drained. Because of conjugated hyperbilirubinaemia cholecystectomy with operative cholangiography (which was normal) were performed. Due to steatotic appearance of the liver a biopsy was performed. Histological examination revealed amyloidosis. The recovery was uneventful and the patient was transferred to hepatology unit for further treatment where she died 3 months later due to gradual deterioration of kidney and heart function. To our knowledge, retroperitoneal haematoma in liver amyloidosis mimicking retropancreatic tumour has not been reported so far.

Clinical, Immunophenotypic and Cytogenetic Features of Megakaryocytic Blast Crisis of Chronic Myeloid Leukemia: A Single Institution Study.

We present 15 patients with megakaryocytic (Mk) blast crisis (BC) of a Philadelphia (Ph) chromosome positive CML confirmed by immunophenotype analysis between 1989-2000. The primary aim of this study is to define clinical, immunological, cytogenetic and laboratory characteristics of Mk BC in Ph positive CML. We have done retrospective analysis regarding basic clinical findings, immunologic phenotype, cytogenetic studies and platelet functions. All patients had significant expression of CD61 (14/14) and CD34 (13/13) antigens, and a high frequency of expression of CD13 (9/12), CD33 (10/12) and CD11b (9/11). The BC in 6/15 patients was presented with thrombocytosis, 7/15 had a normal platelet count and two patients had thrombocytopenia. A grade IV myelofibrosis was present in 8/10 patients. Six patients evolved additional karyotypic abnormalities. Two patients had extramedullary BC. The serum activity of LDH (med. 1095.6) was elevated in all patients. A platelet dysfunction was documented in 4/5 patient tested. There are no clinical and hematological characteristics specific for Mk BC of CML. Normal or elevated platelet count (med. 427.4 x 109/L) in BC of CML with prominent expression of CD34 and CD61 antigens, and significant myelofibrosis (grade IV) are the most consistent clinical findings.

Recurrent venous thrombosis in a patient with chronic lymphocytic leukemia and acquired protein S deficiency.

A patient with chronic lymphocytic leukemia and an undetectable plasma level of protein S (PS), associated with recurrent venous thrombosis, is described. The laboratory investigation revealed the concomitant presence of an inhibitor directed to PS and a monoclonal protein in the patient's plasma. After treatment with prednisone and cyclophosphamide both the inhibitor to PS and the monoclonal component disappeared.

[Hereditary deficiency of antithrombin III, protein C, protein S and factor XII in 121 patients with venous or arterial thrombosis]. / Nasledni nedostatak antitrombina tri, proteina C, proteina S i XII faktora kod 121 bolesnika s venskom ili arterijskom trombozom.

INTRODUCTION: Hereditary thrombophilia is caused by various inherited disorders which lead to familial tendency to recurrent venous thrombosis usually at an early age and with spontaneous onset. In the studies reported so far, the different prevalence of hereditary thrombophilia among patients with venous thrombosis was found, greatly depending on criteria for selection of patients. Arterial thrombosis is most often the consequence of arteriosclerosis but the prevalence of hereditary thrombophilia among young patients with arterial thrombosis and without recognized risk factors for arteriosclerosis is not known . In this study, the frequency of hereditary deficiencies of antithrombin III (AT III), protein C (PC), protein S (PS), plasminogen (PLMG), factor XII (F XII) and dysfibrinogenaemia was investigated over a 2-year period in 121 patients with venous or arterial thrombosis selected according to the recommendations of the British Committee for Standards in Haematology. PATIENTS AND METHODS: The study included total a of 121 patients (58 males and 63 females) with documented venous or arterial thrombosis. Table 1 shows patient's characteristics regarding gender, age and clinical manifestation of thrombosis. Each patient fulfilled at least one of the following criteria: a) venous thrombosis prior to the age of 45; b) arterial thrombosis prior to the age of 30, without risk factors for arteriosclerosis; c) recurrent thrombosis; d) familial tendency to thrombosis; e) thrombosis of unusual localization. A detailed history was taken from each patient on earlier personal or familial occurrence of thrombosis. For the purpose of this study, thrombophilia was characterized as congenital when the deficient protein was constantly below normal value and when the same deficiency was confirmed in a close family member; acquired when the acquired disorder predisposing to thrombosis was present in absence of constant protein deficiency; and idiopathic when the cause of thrombosis was unknown. All tests were performed in plasma obtained after centrifugation of venous blood anticoagulated with 0.129 mol/1 sodium citrate. Concentrations of fibrinogen, PT, PTT and F XII were measured by standard clotting methods. At III, PC and plasminogen activity were determined by chromogenic methods using commercial reagents (Boehring, Marburg, Germany). AT III, PC and total PS antigen were assayed by Laurell immunoelectrophoresis. The presence of lupus anticoagulant was investigated by recommended tests. RESULTS: A total of 15 patients (12.4%) fulfilled criteria for hereditary thrombophilia. Seven of them (5.8%) had AT III deficiency, five (4.1%) PC deficiency, two (1.6%) PS deficiency, and one patient had F XII deficiency. Secondary thrombophilia was found in 21.5% of patients and the cause of thrombosis in 66.1% of patients was not elucidated. A high frequency of hereditary thrombophilia has been found in patients with arterial thrombosis (40%). Among patients with hereditary thrombophilia thrombosis occurred at significantly younger age (29.9 vs. 42.2 and 40.9 yr.) compared to the patients with secondary and idiopathic thrombophilia, respectively. Patients with hereditary thrombophilia had also a higher occurrence of positive family history related to thrombosis (66.7% vs. 7.7% and 27.5%). DISCUSSION: The prevalence of hereditary thrombophilia in nonselected patients with venous thrombosis is relatively low, and for that reason the selection of patients, according recommended criteria, in whom the screening tests for congenital thrombophilia should be performed, is strongly suggested by many authors. In our study we used the generally accepted recommendations for investigation of patients with venous and arterial thrombosis. The presence of congenital thrombophilia was found in 15 (12.4%) of 121 studied patients, what is in accordance with results of other similarly designed studies. (ABSTRACT TRUNCATED)