Neoplasm-induced bleeding in inherited, heterozygous FXIII-A deficiency.

UNLABELLED: Inherited mild factor XIII deficiency belongs to one of the most underdiagnosed bleeding disorders so far. This is, because most patients do not develop bleeding complications in daily life. Patient, methods: A man (age: 64 years) without a history of bleeding presented with painful swelling of neck, weight loss, anemia and episodic bleeding from the right tonsil necessitating tonsillectomy. Histologic and immunohistochemical evaluation revealed cytokeratin-positive epitheloid angiosarcoma. Blood coagulation status showed significantly elevated D-dimer and decreased FXIII levels (FXIII-activity 35%, FXIIIA-Ag 16-26%). Plasma mixing studies excluded neutralizing antibodies against FXIII. RESULTS: A novel heterozygous F13A1 gene nonsense mutation (p.Glu103Ter, c.307G>T) was found confirming heterozygous FXIII-A deficiency. The same mutation was detected in two further asymptomatic relatives. For further clinical management the patient was transfused with FXIII-concentrate and showed an adequate increase of FXIII ruling out FXIII deficiency to be induced by increased turnover. Despite this haemostatic management and antifibrinolytic treatment the patient had to undergo several revisions due to delayed, Hb relevant bleeding after cervical lymph nodes extirpation and resection of tonsil. Two chemotherapy cycles with paclitaxel and palliative radiotherapy of the neck area were performed, but the patient died unfortunately two months after diagnosis. CONCLUSIONS: It is a unique case showing the combination of a highly aggressive angiosarcoma and presence of inherited FXIII deficiency. It is also a rare example demonstrating the benefit of FXIII genotyping besides the expected acquired FXIII deficiency possibly due to neoplasm induced increased consumption by elevated crosslinking of fibrin fibers.

Strong association of variants around FOXE1 and orofacial clefting.

Nonsyndromic orofacial clefting (nsOFC) is a common, complex congenital disorder. The most frequent forms are nonsyndromic cleft lip with or without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO). Although they are generally considered distinct entities, a recent study has implicated a region around the FOXE1 gene in both nsCL/P and nsCPO. To investigate this hypothesis, we analyzed the 2 most strongly associated markers (rs3758249 and rs4460498) in 2 independent samples of differing ethnicities: Central European (949 nsCL/P cases, 155 nsCPO cases, 1163 controls) and Mayan Mesoamerican (156 nsCL/P cases, 10 nsCPO cases, 338 controls). While highly significant associations for both single-nucleotide polymorphisms were obtained in nsCL/P (rs4460498: p Europe = 6.50 × 10(-06), p Mayan = .0151; rs3758249: p Europe = 2.41 × 10(-05), p Mayan = .0299), no association was found in nsCPO (p > .05). Genotyping of rs4460498 in 472 independent European trios revealed significant associations for nsCL/P (p = .016) and nsCPO (p = .043). A meta-analysis of all data revealed a genomewide significant result for nsCL/P (p = 1.31 × 10(-08)), which became more significant when nsCPO cases were added (p nsOFC = 1.56 × 10(-09)). These results strongly support the FOXE1 locus as a risk factor for nsOFC. With the data of the initial study, there is now considerable evidence that this locus is the first conclusive risk factor shared between nsCL/P and nsCPO.

[Anticoagulation]. / Antikoagulation.

Anticoagulant drugs belong to the group of antithrombotic agents and are successfully used in the prophylaxis and treatment of thromboembolic disorders. The use of anticoagulants in the prevention of deep venous thrombosis has significantly lowered the risk of venous thrombosis and fatal pulmonary embolisms even in high-risk situations such as orthopedic surgery. Anticoagulants play a central role in the treatment of acute venous thrombosis and in the prevention of recurrent events. Long-term anticoagulation therapy with orally active anticoagulants significantly reduces the risk of thromboembolic complications in patients showing cardiac arrhythmias. Whereas a few years ago heparins and vitamin K antagonists were the dominant anticoagulants, today a wide range of anticoagulants with improved pharmacological profiles are available. It remains an open question whether these new anticoagulants will improve the efficacy, safety, and acceptance of anticoagulant treatment approaches.

In vitro characterization of recombinant factor VIII concentrates reveals significant differences in protein content, activity and thrombin activation profile.

Recombinant factor VIII (rFVIII) concentrates differ due to cell lines, culture conditions, presence of the B domain and authorized potency assays. This study characterizes three commercially available rFVIII concentrates: a second-generation full length (A), a third-generation full length (B) and a third-generation B domain-deleted (BDD) product (C). rFVIII concentrates were characterized for FVIII activity (FVIII:C) by one-stage clotting and chromogenic assays, FVIII antigen (FVIII:Ag), thrombin activation profile and FXa-generation assay. The rFVIII concentrates exhibited significant differences with regard to FVIII:C, FVIII:Ag and thrombin activation profile. Product A had significantly greater FVIII:C and FVIII:Ag relative to the measured values of products B and C. In addition, product A demonstrated faster and more complete activation by thrombin than the two others. BDD product C had the slowest measured thrombin activation rate. Product A exhibited a greater in vitro FXa generation than products B and C. We found no differences in FXa generation among all three products when FXa generation was normalized for FVIII:Ag. The greater FVIII:C and FVIII:Ag values for product A compared with that for products B and C are due to application of different authorized potency assays (one-stage assay for A vs. chromogenic assay for B and C). The variation in thrombin activation profiles may arise from differences in cell line-dependent posttranslational modifications of the various recombinant proteins.

Monitoring of plasma levels of activated protein C using a clinically applicable oligonucleotide-based enzyme capture assay.

BACKGROUND: Human-activated protein C (APC) is a serine protease with anticoagulant, anti-inflammatory and cytoprotective functions. This feature renders APC to be a promising vascular-inflammatory biomarker. OBJECTIVE: The aim of the present study was the development and validation of a technique that allows the measurement of APC plasma levels under practical laboratory conditions. METHODS/PATIENTS: Based on the APC-binding ssDNA aptamer HS02-52G we developed an oligonucleotide-based enzyme capture assay (OECA) that quantifies aptamer-captured APC through hydrolysis rates of a fluorogenic peptide substrate. After optimization of pre-analytical conditions, plasma APC levels were measured in healthy individuals and patients undergoing hip replacement surgery. RESULTS AND CONCLUSION: A combination of APC-OECA with an aprotinin-based quenching strategy allowed APC analysis with a limit of detection as low as 0.022 ± 0.005 ng mL(-1) (0.39 ± 0.10 pmol L(-1)) and a limit of quantification of 0.116 ± 0.055 ng mL(-1) (2.06 ± 0.98 pmol L(-1)). While APC plasma levels in healthy individuals fell below the quantifiable range of the APC-OECA platform, levels substantially increased in patients undergoing hip replacement surgery reaching peak values of up to 12 ng mL(-1) (214 pmol L(-1)). When normalized to the amount of thrombin generated, interindividual variabilities in the APC generating capacity were observed. In general, with a turn-around time from blood sampling to generation of test results of < 7 h, the APC-OECA platform allows sensitive and rapid determination of circulating APC levels under pathological conditions.

Aptamer-based modulation of blood coagulation.

Nucleic acid based aptamers are single-stranded oligonucleotide ligands isolated from random libraries by an in-vitro selection procedure. Through the formation of unique three-dimensional structures, aptamers are able to selectively interact with a variety of target molecules and are therefore also promising candidates for the development of anticoagulant drugs. While thrombin represents the most prominent enzymatic target in this field, also aptamers directed against other coagulation proteins and proteases have been identified with some currently being tested in clinical trials. In this review, we summarize recent developments in the design and evaluation of aptamers for anticoagulant therapy and research.

Haemostasis management of massive bleeding.

Trauma-induced coagulopathy (TIC) is a frequent complication of severely injured patients. The etiology of TIC is complex. Contributing factors include overwhelming generation of thrombin and activated protein C, consumption of coagulation factors and platelets, hyperfibrinolysis, and dilution of clotting factors through administration of fluids. In addition, hypothermia and shock-associated metabolic acidosis augment the clotting dysfunctions. The occurrence of TIC has been shown to be an independent risk factor for death after trauma warranting aggressive treatment. On admission to the emergency room patients with massive blood loss should be employed on basis of clinical and diagnostic variables to identify patients at high risk of coagulopathy. Patients at high risk should be treated with tranexamic acid (1 g bolus followed by 1 g/8 h), and critical factor and platelet deficiencies should be corrected by transfusion of factor concentrates and platelet concentrates. In addition, plasma should be administered in a 1:1 ratio with red cells. The use of recombinant factor VIIa should be considered if major bleeding persists despite best-practive use of blood products.

Anticoagulant characteristics of HD1-22, a bivalent aptamer that specifically inhibits thrombin and prothrombinase.

BACKGROUND: HD1-22 is a bivalent aptamer that binds to thrombin with high affinity (K(d) = 0.65 nm) and occupies both anion binding exosites without blocking the active centre of the enzyme. HD1-22 has been developed by connecting the exosite 1 binding aptamer HD1 and the exosite 2 binding aptamer HD22 through a poly-dA linker. OBJECTIVES: To characterize the anticoagulant profile of HD1-22 in comparison to the clinically established direct acting thrombin inhibitors bivalirudin and argatroban, and to test the efficacy of antidote-oligodeoxynucleotides. METHODS AND RESULTS: HD1-22 prolongs clotting times of the thrombin time, activated partial thromboplastin time, ecarin clotting time, and lag-time of the tissue factor triggered thrombin generation assay in a dose-dependent manner. On a molar basis, its anticoagulant activity was nearly identical to bivalirudin and superior to argatroban. Thrombin-induced platelet aggregation was more effectively inhibited by HD1-22 than by bivalirudin. The HD1-22 aptamer retains the ability of the HD1-moiety to bind to (pro)exosite 1 of prothrombin and inhibits the prothrombinase activity nearly 2-fold better than HD1. The anticoagulant activities of HD1-22 are fully reversed by addition of antidote-oligodeoxynucleotides. CONCLUSIONS: The strong thrombin-inhibiting activity, together with the availability of a rapid acting antidote strategy, makes HD1-22 an interesting anticoagulant candidate, especially for use in clinical situations where effective anticoagulation and rapid reversal of the anticoagulant effect are required. The data obtained warrant further clinical studies.

[New insight in therapeutic anticoagulation by Coumarin derivatives]. / Neue Einsichten in die orale Antikoagulations-therapie mit Cumarinen.

The recent identification of vitamin K epoxid-reductase complex (VKORC1) contributed significantly to our mechanistic understanding of the vitamin K cycle. VKORC1 protein is targeted by Coumarins. Its enzymatic activity represents the rate-limiting step in the vitamin K cycle and gamma-carboxylation of vitamin K dependent proteins. Possibly, VKORC1 is the only component of VKOR activity. Mutations as well as polymorphisms in coding and non-coding regions of the VKORC1 gene have been shown to cause both partial to total coumarin resistance and coumarin sensitivity. Availability of molecular diagnostics (VKORC1, CYP2C9) and laboratory analysis by HPLC (determination of coumarin, vitamin K and vitamin K epoxide levels) is helpful in detection of hereditary and acquired factors influencing coumarin therapy. In the future, these tools might lead to an individualized and safer oral anticoagulation therapy. Furthermore, daily low-dose vitamin K supplementation may improve stability of coumarin-based anticoagulation. In the perspective of the coming new oral anticoagulants, the efficacy and safety profile of the "old" anticoagulants is of major importance. The well established and oeconomic coumarin drugs will benefit from a pharmacogenetic and nutritive adjusted optimization of therapy.

[The management of patients with heparin-induced thrombocytopenia]. / Management von Patienten mit heparininduzierter Thrombozytopenie.

Heparin-induced thrombocytopenia is a strong risk factor for the development of arterial and venous thromboembolic events. In patients clinically suspected for HIT, immediate cessation of heparin treatment and continuation of anticoagulant treatment using alternative anticoagulants is mandatory in order to minimize the risk of thrombotic events. Alternative anticoagulants that have been successfully used in HIT include the direct acting thrombin inhibitors hirudin, bivalirudin and argatroban, and the heparinoid orgaran. In addition, there is growing evidence that the synthetic pentasaccharide fondaparinux is usable for the treatment of HIT patients. This short review summarizes the strategies of alternate anticoagulant treatment in HIT patients and also describes long-term treatment of HIT patients.

[Biventricular thrombi dissolution and antibody development with lepirudin therapy]. / Biventrikuläre Thrombenauflösung und Antikörper-Bildung unter Lepirudin-Therapie.

HISTORY AND ADMISSION FINDINGS: A 50-year-old patient presented with clinical symptoms of heart failure with orthopnoe and edema (NYHA IV). INVESTIGATIONS: Echocardiography revealed a dilated left ventricle with severely reduced left ventricular function and biventricular floating thrombi, due to dilatative cardiomyopathy. TREATMENT AND COURSE: With a heart failure medication clinical symptoms reduced and body weight decreased > 10 kg in 3 weeks. Due to the high-risk constellation, anticoagulation was performed with lepirudin and the biventricular thrombi were dissolved within 17 days. At this point in time, the patient suffered from petechial bleedings, hemoptysis and gross hematuria. Despite breaking anticoagulation and substitution of PPSB with not measurable fibrinogen, subarachnoid hemorrhage occurred leading to exitus letalis. CONCLUSION: Lepirudin is a highly effective anticoagulant, that can induce severe hemorrhagic side effects in individual cases. The present case report demonstrates an immunological reaction as a rare cause with activation of prothrombin and formation of fibrin.

[Recurrent spontaneous hemorrhage in a patient with light chain immunocytoma]. / Rezidivierende Spontanblutungen bei einer Patientin mit Leichtketten-Immunozytom.

The 48-year-old female patient was sent to our clinic for further evaluation of a spontaneous decrease of prothrombin- and prolongation of the bleeding-time. She presented in good conditions with an enlargement of cervical lymphnodes and the history of a monoclonal plasmacyte dyscrasia. The laboratory results revealed a pronounced decrease of prothrombin-time, a prolonged activated partial thromboplastin-time, a decrease of factor VII and X activity and a light chain paraprotein. The histological examination of the bone marrow led to the diagnosis of an immunocytoma and a medullar amyloidosis. For the aim of influencing the coagulopathy the patient was treated with chemotherapy. However, she developed severe bleedings. Further haemostaseological tests presented an amyloidosis-associated decrease of factor VII and X, an acquired von Willebrands disease, an acquired thrombozytopathy and a lupus-like anticoagulans. Under substitution of factor VIII-von Willebrand-factor-complex and chemotherapeutic treatment a stabilisation over several years was achieved till the patient died due to an amyloid-associated acute pancreatitis.

Screening for thrombophilic risk factors among 25 German patients with cerebral venous thrombosis.

OBJECTIVES: In this study the frequency of inherited thrombophilic risk factors in a population of German CVT patients and their influence on clinical outcome were evaluated. MATERIAL AND METHODS: Twenty-five patients (age 37.1 +/- 16.3 years) with CVT were screened for inherited coagulation disorders. All participants received a full clinical follow-up (mean follow-up period 4.8 +/- 6.4 years). RESULTS: Inherited thrombophilic risk factors were identified in 9 (36%) of the 25 patients studied. Four were found positive for the heterozygous factor V Leiden mutation, 2 were heterozygous carriers of the prothrombin-G20210A-polymorphism. APC resistance proved to be a reliable screening method for factor V Leiden mutation, whereas genetic evaluation for protein S and C deficiencies failed to demonstrate any mutations despite the identification of 1 patient with a protein C and protein S deficiency each. One patient suffered from a familial plasminogen deficiency. These 9 patients had a less favorable outcome (P < 0.05). CONCLUSION: Our results demonstrate that screening for inherited thrombophilia should be an integral part in the diagnostic work up of CVT patients. Patients with inherited coagulopathies tended to have a less favorable outcome, corroborating recommendations for a longer period of oral anticoagulation.

Analysis of size-classified ice crystals by capillary electrophoresis.

In order to analyse the main inorganic cations (NH4+, K+, Na+, Ca2+, Mg2+) and anions (Cl-, NO3-, SO4(2-)) as well as carboxylic and dicarboxylic acids in ice crystals by capillary electrophoresis, electrolyte systems were developed and optimised with respect to limits of detection, resolution, reproducibility and analysis time. We applied indirect UV detection, which enables the simultaneous detection of multiple components. Salicylic acid and 4-methylaminophenolsulfate were used as UV-active co-ions for analysis of anions and cations, respectively. The special features of these systems were low limits of detection in the range 0.3-0.9 micromol L(-1), i.e. absolute limits of detection were in the fmol range, and short analyses times. Separations of cations as well as anions including carboxylic and dicarboxylic acids were completed within 4 min allowing a high sample throughput. Furthermore, the applicability of the newly developed electrolyte systems was demonstrated by comparative analyses with ion chromatography and by first field experimental studies.