The prothrombin 20209 C-->T mutation in Jewish-Moroccan Caucasians: molecular analysis of gain-of-function of 3' end processing.

BACKGROUND: Mutations of the 3' end mRNA-processing signal of the prothrombin (F2) gene have been reported to cause elevated F2 plasma concentrations, thrombosis, and complications of pregnancy. Whereas the common F2 20210*A mutation is almost exclusively found in Caucasians, the F2 20209*T mutation has been reported in Afro-Americans and Afro-Caribbeans only. PATIENTS AND METHODS: Using LightCycler technology, three unrelated Jewish-Moroccan patients tested for obstetric complications were found to be carriers of the F2 20209*T allele. A detailed molecular analysis was performed to identify the functional impact of this mutation. RESULTS: We report three unrelated women of Jewish-Moroccan origin with a F2 20209*T mutation and fetal loss or infertility. The functional analysis revealed that the F2 20209*T mutation stimulates 3' end processing and up-regulates prothrombin protein expression as assessed by a highly sensitive luminescence-based reporter system. CONCLUSIONS: This is the first report of 20209*T in Caucasians, and functional analysis demonstrates that F2 20209*T falls into a general category of mutations of the F2 gene, which may possibly contribute to thrombophilia and complications of pregnancy by interfering with a tightly balanced architecture of non-canonical F2 3' end formation signals.

Recombinant activated factor VII (NovoSeven): addition to replacement therapy in acute, uncontrolled and life-threatening bleeding.

BACKGROUND AND OBJECTIVES: Recombinant activated factor VII (rFVIIa, NovoSeven) has been used off-label for various conditions. A protocol for its use in acute, uncontrolled life-threatening bleeding, was devised and employed. A haematologist/transfusion specialist was assigned as a member of the team. MATERIALS AND METHODS: The clinical data were reviewed and summarized. A scoring system for the assessment and monitoring of coagulopathy was employed. Each parameter of prothrombin time (PT), activated partial thromboplastin time (aPTT), platelet number and fibrinogen level was allocated points according to the degree of abnormality. Three scoring levels emerged. RESULTS: Between April 2001 and April 2003, 13 patients received rFVIIa for acute, uncontrolled life-threatening bleeding. Nine of 13 patients remained alive for 15 days or longer after rFVIIa infusion. All patients who experienced a reduction or cessation of bleeding after rFVIIa infusion, also had a lower coagulopathy score after replacement therapy, prior to rFVIIa infusion, compared with their score at rFVIIa request. There was a reduction in the average use of blood products after rFVIIa infusion. The coagulopathy score was statistically predictive of response to rFVIIa and survival. CONCLUSIONS: In an area where very little data exists, we report the usefulness of rFVIIa. We propose that transfusion replacement should aim to correct coagulopathy before infusion of rFVIIa and that a haematologist/transfusion specialist should be involved in the management of these patients. A prognostically significant coagulopathy scoring system is offered.

Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases.

Myeloablative conditioning associated with hazardous immediate and late complications is considered as a mandatory first step in preparation for allogeneic blood or marrow transplantation (allogeneic BMT) for the treatment of malignant hematologic disorders and genetic diseases. Immune-mediated graft-versus-leukemia (GVL) effects constitute the major benefit of allogeneic BMT. Therefore, we have introduced the use of relatively nonmyeloablative conditioning before allogeneic BMT aiming for establishing host-versus-graft tolerance for engraftment of donor immunohematopoietic cells for induction of GVL effects to displace residual malignant or genetically abnormal host cells. Our preliminary data in 26 patients with standard indications for allogeneic BMT, including acute leukemia (n = 10); chronic leukemia (n = 8), non-Hodgkin's lymphoma (n = 2), myelodysplastic syndrome (n = 1), multiple myeloma (n = 1), and genetic diseases (n = 4) suggest that nonmyeloablative conditioning including fludarabine, anti-T-lymphocyte globulin, and low-dose busulfan (8 mg/kg) is extremely well tolerated, with no severe procedure-related toxicity. Granulocyte colony-stimulating factor mobilized blood stem cell transplantation with standard dose of cyclosporin A as the sole anti-graft-versus-host disease (GVHD) prophylaxis resulted in stable partial (n = 9) or complete (n = 17) chimerism. In 9 patients absolute neutrophil count (ANC) did not decrease to below 0.1 x 10(9)/L whereas 2 patients never experienced ANC < 0.5 x 10(9)/L. ANC > or = 0.5 x 10(9)/L was accomplished within 10 to 32 (median, 15) days. Platelet counts did not decrease to below 20 x 10(9)/L in 4 patients requiring no platelet support at all; overall platelet counts > 20 x 10(9)/L were achieved within 0 to 35 (median 12) days. Fourteen patients experienced no GVHD at all; severe GVHD (grades 3 and 4) was the single major complication and the cause of death in 4 patients, occurring after early discontinuation of cyclosporine A. Relapse was reversed by allogeneic cell therapy in 2/3 cases, currently with no residual host DNA (male) by cytogenetic analysis and polymerase chain reaction. To date, with an observation period extending over 1 year (median 8 months), 22 of 26 patients (85%) treated by allogeneic nonmyeloablative stem cell transplantation are alive, and 21 (81%) are disease-free. The actuarial probability of disease-free survival at 14 months is 77.5% (95% confidence interval, 53% to 90%). Successful eradication of malignant and genetically abnormal host hematopoietic cells by allogeneic nonmyeloablative stem cell transplantation represents a potential new approach for safer treatment of a large variety of clinical syndromes with an indication for allogeneic BMT. Transient mixed chimerism which may protect the host from severe acute GVHD may be successfully reversed postallogeneic BMT with graded increments of donor lymphocyte infusions, thus resulting in eradication of malignant or genetically abnormal progenitor cells of host origin.

High-dose dexamethasone for splenectomy in patients with idiopathic thrombocytopenic purpura.

High-dose intravenous immune globulin (IV IgG) is currently the treatment of choice for patients with idiopathic thrombocytopenic purpura (ITP) who undergo splenectomy; however, this treatment is extremely expensive. We report on 13 ITP patients with severe thrombocytopenia (<20 x 10(9)/l) who were prepared for laparoscopic splenectomy with a 4-day oral course of high-dose (40 mg/day) dexamethasone (DEX). Four patients had an excellent response with platelet counts that increased to above 150 x 10(9)/l. Seven patients had a good response with a platelet count that increased to between 50 and 150 x 10(9)/l (median 121 x 10(9)/l). Two patients were resistant both to DEX and IV IgG. The operation was uneventful in all the patients, including the 2 who had resistant ITP and were operated on while their platelet count was very low (5 x 10(9)/l). Thus, high-dose DEX, which is an easy, effective and inexpensive treatment, is recommended for the preparation of ITP patients prior to splenectomy.

Highly elevated lactate dehydrogenase level in a healthy individual: a case of macro-LDH.

Macroenzymes are complexes of serum enzymes with a plasmatic protein. They have a higher molecular weight and a more prolonged serum half-life than those of unbound enzymes. Although macroenzymes may be found in the serum of post-myocardial infarction patients, they are not usually associated with any specific disease. Their presence, however, can cause an elevation in the serum levels of an enzyme, possibly leading to errors in diagnosis. We report a patient with extremely elevated serum levels of lactate dehydrogenase (LDH) due to the formation of complexes with immunoglobulin G. She had undergone a myriad of clinical examinations until the macroenzyme responsible for this finding was detected. We also review the literature on the clinical significance of macro-LDH. We propose that awareness of this rare and probably benign phenomenon can spare the patient from the distress of exhaustive investigations.

[Macroenzymes: an interesting laboratory finding, without clinical relevance].

Macroenzymes are complexes of serum enzymes with proteins which have a higher molecular weight and longer plasma half-life than the normal enzyme. The presence of macroenzymes is suggested by finding increased serum enzyme activity, not associated with symptoms. Thus, macroenzymes can cause diagnostic errors and the performance of unnecessary tests or invasive procedures. We describe 2 patients with highly elevated serum levels of lactate dehydrogenase (LDH) and creatine kinase (CK) due to formation of complexes with immunoglobulin G. 1 patient had LDH of 4500 u/L but was otherwise normal and in the second CK was elevated with no evidence of ischemic heart disease. Awareness of the phenomenon of macroenzymes may save the patient long and sometimes invasive investigation.

[Pulsed high-dose dexamethasone in resistant immune thrombocytopenic purpura].

Most patients with chronic idiopathic thrombocytopenic purpura (TTP) show a good initial response to treatment with corticosteroids. However the disease relapses in more than 90% when steroid dosage is reduced. Recently 100% success was reported for a new therapeutic protocol in 12 patients (ranging in age from 13-60, half of them women) with chronic ITP refractory to corticosteroids or to splenectomy. They were given pulsed therapy with oral dexamethasone, 40 mg/day on 4 consecutive days each month, for 6 months. This treatment protocol was used in an attempt to avoid splenectomy. 5 patients (42%) had a complete response but 7 did not. The median follow-up in those who responded was 7 months (range 6-8). Of the 7 who did not respond, 5 had not completed treatment: 3 because of urgent splenectomy and 2 because of lack of response after 3 courses of therapy accompanied by side-effects. Most patients suffered typical corticosteroid side-effects, principally restlessness, insomnia, and withdrawal effects. These were milder and better tolerated in those treated with Dexacort solution (20 mg ampules) rather than dexamethasone tablets. Despite complete response in only 5 of the 12 patients (42%), we feel that pulsed high-dose dexamethasone is effective and should be tried in TTP refractory to conventional corticosteroid therapy, before resorting to splenectomy.

Construction, expression, and characterization of a chimera of factor IX and factor X. The role of the second epidermal growth factor domain and serine protease domain in factor Va binding.

The prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin, consists of activated Factor X, Factor Va, a membrane surface and Ca2+. To examine the structures that support Factor Va binding to Factor X, we used in vitro mutagenesis to construct a chimeric molecule that includes regions of Factor IX and Factor X. This chimera (IXGla,E1XE2,SP) was prepared from cDNA encoding the second epidermal growth factor (EGF) and serine protease domains of Factor X linked downstream from the cDNA encoding the signal peptide, propeptide, Gla domain, and first EGF domain of Factor IX. The cDNAs encoding the Factor IX/X chimera and wild-type Factor X were each expressed in Chinese hamster ovary cells and the secreted proteins purified by affinity chromatography using polyclonal anti-Factor X antibodies. The chimera migrated as a single major band corresponding to a molecular weight of 68,000. By Western blotting, the chimeric protein stained with both polyclonal anti-Factor X and anti-Factor IX antibodies. gamma-Carboxyglutamic acid analysis demonstrated near complete carboxylation of both the wild-type Factor X and the Factor IX/X chimera. Compared with Factor X, the rate of zymogen activation of the Factor IX/X chimera was about 50% that of Factor X when activated by Factor IXa, Factor VIIIa, phospholipid, and Ca2+. The enzyme form of the Factor IX/X chimera, activated Factor IX/X, generated using the coagulant protein of Russell's viper venom, expressed full amidolytic activity compared with Factor Xa. The activated Factor IX/X chimera had about 14% of the activity of Factor Xa when employed in a prothrombinase assay; this activity reached 100% with increasing concentrations of Factor Va. A binding assay was employed to test the ability of the active site-inactivated Factor IX/Xa chimera to inhibit the binding of Factor Xa to the Factor Va-phospholipid complex, thus inhibiting the activation of prothrombin to thrombin. In this assay the active site-inactivated form of the chimera competed with Factor Xa completely but with decreased affinity for the Factor Va-phospholipid complex. These data indicate that the second EGF domain and the serine protease domain of Factor Xa are sufficient to interact with Factor Va. The Factor IX/X chimera is a good substrate for the tenase complex; the defective enzymatic activity of the activated Factor IX/X chimera can be accounted for by its decreased affinity for Factor Va relative to Factor Xa.

The relative frequency of hereditary thrombotic disorders among 107 patients with thrombophilia in Israel.

Since most patients with thrombophilia in Israel are referred for diagnosis to our center, it was possible to estimate the relative frequency of the hereditary disorders leading to thrombophilia. 107 unrelated patients were evaluated over 4 years. Diagnoses were established in 23 patients (21.5%) while in 84 (78.5%) no abnormality was detected. Antithrombin III deficiency was found in 8 patients (7.5%), dominant protein C deficiency in 6 (5.6%), recessive homozygous protein C deficiency in 1, protein S deficiency in 3 (2.8%) and dysfibrinogenemia in 1. Four additional patients (3.7%) had a lupus anticoagulant. The frequency of deep vein thrombosis and pulmonary embolism was similar in patients with and without a definite diagnosis. Thrombosis of visceral or cerebral vessels and a positive family history were more frequent among patients in whom a definite diagnosis was made. In both groups there was a substantial lag between the time of presentation of the first thrombotic episode and the time of evaluation. Since the number of referred patients with thrombophilia has gradually increased over the period of the study, it is at present impossible to establish the prevalence of the various hereditary disorders leading to thrombophilia in the population.

The outcome of bacterial infection in subjects with benign familial leukopenia (BFL).

Benign familial leukopenia (BFL) is a hereditary phenomenon, encountered in several ethnic groups. Subjects bearing BFL are believed to be affected by bacterial infection in no greater incidence than normal subjects. In our study we investigated a group of subjects with BFL during an acute bacterial infection in comparison to subjects without BFL with the same infection. We found that the subjects with BFL had no absolute leukocytosis during the infection. Nevertheless, they reacted similarly to the other subjects in regard to their temperature and heart rate; however, they were hospitalized for fewer days than subjects without BFL. We conclude that BFL is a benign phenomenon, requiring neither specific treatment as such, nor more aggressive therapy during infection. The benign course of an acute bacterial infection in BFL indicates that perhaps the number of WBC's that are normally recruited during an infection in normal subjects highly exceeds that which is necessary.