In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

BACKGROUND: Congenital afibrinogenemia is a rare inherited autosomal recessive disorder in which a mutation in one of three genes coding for the fibrinogen polypeptide chains Aα, Bß and γ results in the absence of a functional coagulation protein. A patient with congenital afibrinogenemia, resulting from an FGA homozygous gene deletion, underwent an orthotopic liver transplant that resulted in complete restoration of normal hemostasis. The patient's explanted liver provided a unique opportunity to further investigate a potential novel treatment modality. OBJECTIVE: To explore a targeted gene therapy approach for patients with congenital afibrinogenemia. METHODS AND RESULTS: At the time of transplant, the patient's FGA-deficient hepatocytes were isolated and transduced with lentiviral vectors encoding the human fibrinogen Aα-chain. FGA-transduced hepatocytes produced fully functional fibrinogen in vitro. CONCLUSIONS: Orthotopic liver transplantation is a possible rescue treatment for failure of on-demand fibrinogen replacement therapy. In addition, we provide evidence that hepatocytes homozygous for a large FGA deletion can be genetically modified to restore Aα-chain protein expression and secrete a functional fibrinogen hexamer.

Bilateral periorbital ecchymoses. An often missed sign of amyloid purpura.

Immunoglobulin light chain (AL) amyloidosis is a systemic disease caused by a plasma cell clone synthesizing an unstable light chain, which forms amyloid fibrils. Deposition of amyloid fibrils affects primarily kidney, heart, nervous system, spleen, liver, gastrointestinal tract and the skin. Skin bleeding in these patients is called amyloid purpura. Classically, it occurs spontaneously and bilaterally in the periorbital region. Vessel wall fragility and damage by amyloid are the principal causes of periorbital and gastrointestinal bleeding. Additionally, coagulation factor inhibitory circulating paraprotein, hyperfibrinolysis, platelet dysfunction or isolated acquired factor X deficiency may contribute to even more severe, diffuse bleedings. Early diagnosis remains essential for improving prognosis of patients with AL amyloidosis. Although pictures of amyloid purpura have been often reported in the literature, the clinical diagnosis may be delayed. We report a case of cutaneous manifestation of AL amyloidosis diagnosed not until one year after the appearance of the first symptoms. Diagnostic work-up revealed that the patient suffered from multiple myeloma with secondary AL amyloidosis. Atraumatic ecchymoses at the face, particularly the eyelids as well as in the neck should raise the suspicion of AL amyloidosis.

Life threatening bleeding under adequate oral anticoagulation. Cases 4a, b.

We report on two men with severe and recurrent bleeding episodes under well performed phenprocoumon therapy. Both patients showed an INR value within the therapeutic/subtherapeutic range with an inappropriately prolonged activated partial thromboplastin time (aPTT). The vitamin K-dependent coagulation factors were in the expected range, except for factor IX, which was as low as in moderate haemophilia B. After substitution of vitamin K, factor IX and the aPTT recovered completely in one case. In the other case factor IX was not measured but aPTT normalized. Diagnosis of an increased sensitivity of factor IX to phenprocoumon was assumed and proven in both cases by the demonstration of a missense mutation at ALA-10 in the factor IX propeptide.

Venous thrombosis and multiple prothrombotic gene defects. Case 5.

We describe the case of a 30-year-old woman with homozygous prothrombin G20210A transition and heterozygous FV Leiden mutation and a history of postpartum venous thrombosis. Despite the high thrombotic risk to be assumed in the presence of this combined thrombophilia our patient suffered her first venous thrombotic event only at the age of 29 years during the puerperium of her first pregnancy. This fact supports the concept of venous thrombosis as a multicausal disease, with interaction of genetic and acquired risk factors. The case presented also stresses the importance of performing complete thrombophilia investigation.

Recurrent thrombosis despite anticoagulation in a man with monocytosis. Case 8.

Recurrent thromboembolism despite oral anticoagulation is primarily suspicious of overt or occult neoplasia. We report on a man (age: 67 years) who presented with severe thrombophilia which was only controlled when the patient was set on a combined anticoagulation with low molecular weight heparin in supratherapeutic dosage and phenprocoumon with a target INR of 2.0. Despite repeated evaluation over about two years, a malignant tumour could never be demonstrated. However, the patient suffered in addition to a protein S deficiency from an antiphosphospholipid syndrome and a chronic myelomonocytic leukaemia. We postulate that the accepted strong thrombogenicity of antiphosphospholipid syndrome was further increased by protein S deficiency and a possibly procoagulatory effect of the abnormal monocytes explaining the severe thrombophilia resistant to standard therapeutic anticoagulation with a vitamin K antagonist and usual therapeutic doses of low molecular weight heparin, respectively.

Hereditary spherocytosis and hemochromatosis.

A 37-year-old male, splenectomized at the age of 1 year, was admitted to the ward with severe chest pain and signs of cardiogenic shock. Clinical investigations revealed the presence of both hemochromatosis and hereditary spherocytosis (HS). HLA typing showed A3,B7 and A24,B57 haplotypes and genetic analysis revealed homozygosity for the C282Y mutation. A family study was performed. The parents and four brothers were heterozygous for the C282Y mutation. Two of the brothers also presented high levels of iron stores and they had been splenectomized because of HS, while two other siblings had neither spherocytosis nor hemochromatosis. The mother had a mild anemia with dehydrated red blood cells (RBC), while the father appeared to have low-density, but normal RBC; none of them presented with spherocytosis. All siblings with spherocytosis and elevated iron stores showed a RBC density distribution similar to the mother. We present the first case with genetically proven hemochromatosis in combination with spherocytosis, focusing on the various possibilities of iron accumulation in individuals with spherocytosis and heterozygosity for the C282Y mutation.

[Recurrent thromboembolisms despite oral anticoagulation in a 76-year-old patient--Trousseau syndrome]. / Rezidivierende Thromboembolien trotz oraler Antikoagulation bei einem 76jährigen Patienten--Trousseau Syndrom.

We report the case of a 76-year-old man with recurrent thromboses and low-grade chronic disseminated intravascular coagulation despite therapeutic oral anticoagulation with phenprocoumon. Work-up revealed a bronchial carcinoma (NSCCL) with hilar and mediastinal lymph node metastasis. The clinical condition was consistent with Trousseau's syndrome. Based on reports in the literature, the therapy was changed from phenprocoumon to intravenous unfractionated heparin (UFH), which was effective in controlling the thrombotic coagulopathy. For practical reasons, despite lack of established effectiveness in Trousseau's syndrome, therapy was switched to low-molecular-weight heparin (LMWH, nadroparine) in therapeutic dosage of 100 IU/kg body wt. subcutaneously 12 hourly. The patient remained free from further thromboembolic events during the last 6.5 months of his life. This case suggests that LMWH might be a convenient alternative to the established therapy with UFH in Trousseau's syndrome.

[A 26-year-old woman with splanchnic vein thrombosis as the initial manifestation of polycythemia vera]. / Eine 26jährige Frau mit splanchnischer Venenthrombose als Initialmanifestation einer Polycythaemia vera.

A 26-year-old woman, after cesarean section in the 33rd week of gestation, developed after delivery thrombosis of the popliteal vein, pulmonary embolism and thrombosis of the portal vein. After completion of a six month period of oral anticoagulation, laboratory investigations revealed diminished levels of plasminogen and free protein S antigen as well as APC-resistance due to heterozygous FV R506Q mutation. After six uneventful years, abdominal sonography and magnetic resonance examination, performed because of abdominal pain, showed liver cirrhosis with Budd-Chiari syndrome. Additional hematological investigations led to the diagnosis of polycythemia vera. Association of myeloproliferative disorders, mainly polycythemia vera, with splanchnic venous thrombosis is well known and should always be looked for.

[32-year-old patient with systemic lupus erythematosus, thrombocytopenia and ischemic cerebrovascular insult and antiphospholipid antibodies]. / 32jährige Patientin mit systemischem Lupus Erythematodes, Thrombozytopenie und ischämischem zerebrovaskulärem Insult sowie Antiphospholipid-Antikörpern.

We report a young woman suffering from systemic lupus erythematosus complicated by severe thrombocytopenia and high-level antiphospholipid antibodies. Thrombocytopenia was refractory to any therapy. In the course of the illness the patient developed an ischemic cerebrovascular stroke. We discuss the clinical features of the antiphospholipid antibody syndrome being primarily characterized by recurrent thromboembolism and address the appropriate prophylactic measures to avoid recurrent events.

Performance of a new fibrin monomer assay to exclude deep vein thrombosis in symptomatic outpatients.

UNLABELLED: In this study we prospectively assessed the reliability of a new fibrin monomer assay in 106 outpatients with clinically suspected deep venous thrombosis of the lower limb. According to the results of the objective tests and using different cut-off points we calculated the sensitivity, specificity and negative predictive value of the fibrin monomer assay. The prevalence of deep vein thrombosis was 44.3% (31.1% proximal, 13.2% distal). Using a cut-off level of plasma fibrin monomer of 3.5 microg/ml, a sensitivity, specificity and negative predictive value of 100% (95% CI: 94-100%), 35.6% (95% CI: 23-48%) and 100% (95% CI: 86-100%), respectively, were obtained. The exclusion rate was 19.8% (95% CI: 12-27%) of all referred patients. These accuracy indices compared favourably with the respective results of a routine D-dimer ELISA used for comparison. CONCLUSION: This new fibrin monomer assay appears to be a reliable method for the exclusion of deep vein thrombosis in symptomatic outpatients.

[Comparison of Quick/INR values of whole capillary blood (CoaguChek Plus) and venous citrate plasma in patients with and without oral anticoagulation]. / Vergleich der Quick-/INR-Werte aus kapillärem Vollblut (CoaguChek Plus) und venösem Citratplasma bei Patienten mit und ohne orale Antikoagulation.

BACKGROUND: The prothrombin time, also called thromboplastin time ("Quick"), is usually measured by using citrated plasma from venous blood. Recently, portable coagulation monitors have been developed which measure prothrombin time using non-anticoagulated capillary whole blood from a finger-stick. In the present study we evaluated the CoaguChek Plus coagulation monitor in comparison with a standard laboratory method in various patient groups: patients on oral anticoagulation with or without heparinisation, patients receiving heparin without oral anticoagulation, patients with a deficiency of one of the coagulation factors of the extrinsic or common pathway, and patients with liver disease. Furthermore, we studied the influence of the haemoglobin concentration on the test results. METHOD: Capillary prothrombin time was measured by using the portable coagulation monitor CoaguChek Plus and venous prothrombin time was assessed by using Thromborel S. RESULTS: We found a correlation coefficient of 0.94 between capillary and venous INR values in 216 determinations from 167 patients. The slope of the regression line was 1.03, and the y-intercept 0.05, 93.5% of the results were within 0.9, 90.7% within 0.7, and 83.8% within 0.5 INR units. Similar results were obtained in patients on oral anticoagulation, patients with a deficiency of a factor of the extrinsic system and in patients with liver disease. Correlation and agreement were somewhat lower among patients on oral anticoagulation and simultaneous heparinisation (40 patients): correlation coefficient was 0.83, slope of the regression line was 0.87 and y-intercept was 0.27 INR units. No influence of the haemoglobin concentration on INR results could be demonstrated. CONCLUSION: Our results show the CoaguChek Plus coagulation monitor to be a valuable tool for measuring prothrombin time in patients on oral anticoagulation, in patients with liver disease to estimate the capacity of protein synthesis, and to screen for possible deficiencies of one of the coagulation factors of the extrinsic or common pathway. However, based on our preliminary data we cannot recommend the use of the CoaguChek Plus coagulation monitor in heparinised patients.

[Ambulatory therapy of acute deep thrombophlebitis]. / Ambulante Therapie der akuten tiefen Beinvenenthrombose.

A 53-year-old man developed deep vein thrombosis seven days after arthroscopic meniscectomy. He was successfully treated at home with a low-molecular-weight heparin followed by oral anticoagulation. Today, low-molecular-weight heparins are the initial treatment of choice for acute deep vein thrombosis. Due to the simplicity of administration, they have the potential for allowing home treatment. Several recently published randomised and controlled studies have demonstrated the feasibility, safety and efficacy of home treatment.

Is plasminogen deficiency a thrombotic risk factor? A study on 23 thrombophilic patients and their family members.

The role of plasminogen (plg) deficiency in the pathogenesis of venous thromboembolism is debated in the literature. In the present study we evaluated the prevalence of plg deficiency in our thrombophilia patients and aimed to elucidate the thrombosis risk of plg deficiency as a single defect or in combination with other defects, with special focus on APC resistance. The study cohort included 1192 consecutive patients with a history of clinically or objectively diagnosed venous and/or arterial thromboembolism and/or positive family history who were referred to our department for thrombophilia investigation from 02/1988 to 03/1997. All available family members of patients with plg deficiency were tested for plg, APC resistance and other thrombophilic defects that were established in the propositus. 23/1192 propositi were plg-deficient corresponding to an overall prevalence of 1.9%, i.e. 2.2% in patients with venous thrombosis and 1.4% in those with arterial events. Out of the 23 plg-deficient propositi, 8 showed one or multiple additional thrombophilic defects, and in 4 patients relevant circumstantial risk factors were present. Of the 53 available family members, 28 were plg-deficient including 5 with additional APC resistance, and 4 subjects had isolated APC resistance. Ten of the 53 family members had already suffered thromboembolic events, i.e. 5 (18%) in the plg-deficient group and 5 (20%) in the non-deficient group, both groups showing an almost identical median age at the time of investigation (28.9 years and 27.1 years, respectively). Based on our data, plg deficiency is a rare defect in thrombophilic patients and as a single defect it does not seem to be a strong thrombotic risk factor, as 11 of 23 propositi had additional thrombophilic defects or circumstantial risk factors, and in the family members thrombotic events were equally frequent in the plg-deficient and non-deficient subjects.

Subcutaneous low-molecular-weight heparin for treatment of Trousseau's syndrome.

We report the case of a 76-year-old man with recurrent thromboses despite oral anticoagulation with phenprocoumon and low-grade chronic disseminated intravascular coagulation. Workup revealed a bronchial carcinoma (NSCCL) with hilar and mediastinal lymph node metastases. The clinical condition was consistent with Trousseau's syndrome. Based on reports in the literature, the therapy was changed to intravenous unfractionated heparin (UFH), which was effective in controlling the thrombotic coagulopathy. For practical reasons, despite a lack of evidence of its effectiveness in Trousseau's syndrome, therapy with UFH was changed to subcutaneous low-molecular-weight heparin (LMWH, nadroparine) in therapeutic doses of 100 IU/ kg body wt. 12 hourly. On an outpatient basis, five chemotherapy cycles were administered, and after metastases of the brain had been detected radiotherapy was initiated. Following 7.5 months of progressive neoplastic disease the patient died. He had remained free of thromboembolic complications under continued LMWH therapy during the last 6.5 months of his life. LMWH might be a convenient alternative to the established therapy with UFH in Trousseau's syndrome.

[The significance of APC resistance (activated protein C) for clinical practice]. / Bedeutung der APC-Resistenz für die Praxis.

Resistance against the anticoagulatory effect of activated protein C (APC resistance) is the most prevalent hereditary risk factor for venous thromboembolism known today. In this short review, an exemplary casuistic observation from our thrombophilia outpatient ward is presented. The discovery by Dahlbäck et al. in 1993 of hereditary APC resistance as a basis of familial thrombophilia and the molecular characterization of this defect as point mutation in the coagulation factor V gene (FV R506Q- or FV Leiden-mutation) by Bertina and coworkers are discussed. The clinical implications of this thrombophilia and relevant knowledge for daily practice are briefly summarized.

[Contribution of the hemostasis laboratory in the diagnosis of deep venous thrombosis]. / Beitrag des Hämostaselabors bei der Diagnostik der tiefen Venenthrombose.

Venography is still the gold standard for objective diagnosis of deep venous thrombosis (DVT). However, this investigation is costly and time-consuming, demands technical expertise and has potential side-effects. To reduce the number of phlebographies a simple, rapid and reliable screening test for exclusion of DVT would be greatly appreciated. In many studies markers of activated coagulation and fibrinolysis such as thrombin-antithrombin complex (TAT), prothrombin fragment 1 + 2 (F 1 + 2) and D-dimers, respectively, were investigated with respect to their potential use as screening tests. Especially D-dimers as assessed by ELISA have turned out to have high sensitivity and negative predictive value for DVT and could therefore serve as screening test for exclusion of DVT. However, most of the easier, more rapid D-dimer Latex tests which would be better suited for emergency situations as well as the TAT- and F 1 + 2-ELISA tests are according to most studies not sufficiently sensitive. In the present article the significance of the different activation markers in the diagnostic approach of DVT is discussed based on the literature.

No association of APC resistance with myocardial infarction.

Resistance to activated protein C (APC resistance) due to the factor V mutation 506 Arg-->Gln (factor V Leiden) is the most prevalent inherited risk factor for venous thromboembolism. Its association with arterial thromboembolic disease, however, is still controversial. In the present study we found no difference between the prevalence of APC resistance (assessed by the ratio of the aPTT with and without added APC) in 134 non-anticoagulated survivors of myocardial infarction and that in 100 controls of similar age and sex distribution (2.2% and 2.0%, respectively). Patients showed a significantly higher median value for the aPTT ratio than controls (2.85 and 2.66, respectively), a fact we could not explain by our data.

[Evaluating the origin of thrombophilia: indications and implementation]. / Thrombophilieabklärung: Indikation und Durchführung.

The overall incidence per year of deep vein thrombosis is about one per thousand, but may be much higher in the presence of certain clinical risk factors such as advanced age, immobilization, surgical procedures, pregnancy, puerperium, use of oral contraceptive agents and malignancy. Moreover, homocystinuria, nephrotic syndrome, systemic lupus erythematosus and hematological disorders such as paroxysmal nocturnal hemoglobinuria or myeloproliferative syndromes predispose to thrombotic disease. Evaluation of the patient with thromboembolism should include detailed history, clinical examination and laboratory investigation to exclude these secondary thrombophilic states. Primary or hereditary thrombophilia is suspected mainly in patients suffering from (venous) thromboembolism at an early age (< 45 years), especially if recurrent and/or familial thrombosis is present. Hereditary thrombophilia may be due to deficiency of antithrombin III, protein C, protein S or plasminogen, some other defects being less well-established prethrombotic risk factors. These currently recognized primary prethrombotic molecular defects are found in 10 to 30% of patients with idiopathic thromboembolism. In the majority of cases the cause of thrombosis remains unknown.