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Acquired hemophilia A (AHA), a rare bleeding disorder caused by neutralizing autoantibodies against coagulation factor VIII (FVIII), occurs in both men and women without a previous history of bleeding. Patients typically present with an isolated prolonged activated partial thromboplastin time due to FVIII deficiency. Neutralizing antibodies (inhibitors) are detected using the Nijmegen-modified Bethesda assay. Approximately 10% of patients do not present with bleeding and, therefore, a prolonged activated partial thromboplastin time should never be ignored prior to invasive procedures. Control of acute bleeding and prevention of injuries that may provoke bleeding are top priorities in patients with AHA. We recommend treatment with bypassing agents, including recombinant activated factor VII, activated prothrombin complex concentrate, or recombinant porcine FVIII in bleeding patients. Autoantibody eradication can be achieved with immunosuppressive therapy, including corticosteroids, cyclophosphamide and rituximab, or combinations thereof. The median time to remission is 5 weeks, with considerable interindividual variation. FVIII activity at presentation, inhibitor titer and autoantibody isotype are prognostic markers for remission and survival. Comparative clinical studies to support treatment recommendations for AHA do not exist; therefore, we provide practical consensus guidance based on recent registry findings and the authors’ clinical experience in treating patients with AHA.

Setting In the article, we remember the role of antithrombin (AT) in hemostasis, escalation of AT-potential with heparin and difficulties with monitoring the effectiveness of LMWH therapy (low molecular weight heparin) in patients with AT deficiency. We pay most of our attention to hereditary AT deficiency and its thromboembolic risk in pregnancy. Methods In the introduction, the principle of AT function, its two main domains and the regulation of synthesis are cleared. We describe the causal mutations of hereditary AT deficiency in SERPINC1 gen and the relation to a thromboembolic risk. The general recommendations for patients with hereditary AT deficiency and pregnant women are mentioned. As the risk of thromboembolic disease is escalated in pregnancy, the LMWH should always be considered. There has been frequently observed that patients with AT deficiency do not elevate anti-Xa-levels when standard prophylactic LMWH doses are used. This fact well illustrates that heparin without AT may not inhibit the active coagulant factors efficiently enough. Therefore, if a high thromboembolic risk in the patients anamnesis is present, the LMWH dosing should be escalated. In individual cases, concomitant administration of an antithrombin concentrate to the heparin treatment is recommended at the time of delivery or in the case of deep venous thrombosis. In this article, three cases of unusual pregnancy in patients with different types of AT deficiency are reported. The case reports are summarized from the Department of Hematology at Hospital Kolin, the Centre of Hemostasis and Thrombosis at Institute of Hematology and Blood Transfusion in Prague and from cooperating obstetrical departments in the Czech Republic. Results We demonstrated the threat of hereditary AT deficiency in three case reports. In one case, the estimated risk of thromboembolism - type I of AT-deficiency (quantitative) - was in a good correlation with real peripartal complications. In the next two cases with different types of AT deficiency, we showed surprising courses of complicated pregnancies. Conclusion As it has been shown, it is not safe to estimate the risk of thromboembolism on the base of causal mutation for AT deficiency. For present clinical practice, we should still remember AT deficiency as a potentially very dangerous thromboembolic disorder for mother and fetus; thus, excellent cooperation of an obstetrician and a hematologist is necessary. Publication ethics: The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for bio medical papers. Conflict of interests: The authors declare they have no potential conflicts of interest concerning the drugs, products or services used in the study. Acknowledgment: I hereby thank Peter Salaj, MD (the current head of the Center for Thrombosis and Hemostasis of the Institute of Hematology and Transfusion (UHKT) in Prague) for inspiring comments on the creation of the article. I also thank the entire Center for Thrombosis and Hemostasis team and the head of the National Reference Laboratory for Hemostasis Disorders at UHKT in Prague, RNDr. Ingrid Hrachovinova, PhD. My thanks also go to the head of the Transfusion and Hematology Department in Kolin, Dagmar Chalupova, MD, and to her team.

INTRODUCTION Acquired haemophilia A (AHA) is a rare autoimmune disorder, characterized by bleeds of varying severity caused by autoantibodies against factor VIII (FVIII). AIM Identify risk factors associated with AHA-related deaths/relapses and assess the effect of increased corticosteroid doses. METHODS AHA patients treated across two specialist centres in the Czech Republic, generally receiving first-line haemostatic therapy with rFVIIa and immunosuppression with corticosteroids/cyclophosphamide, were included. We analysed the association between early death (within 8 weeks of diagnosis [considered disease-related]) and age, malignancy, FVIII levels and bleeding severity. Risk factors associated with reduced 2-year survival and relapse incidence, and the effect of increased corticosteroid doses on early death and remission were also assessed. RESULTS The demographics of the described cohort (n = 66) were similar to other AHA registries. Early death occurred in 20% of cases. Unlike age and malignancy, FVIII levels

Hemostasis can be characterized as an array of physiological mechanisms providing both blood fluidity in the intact blood vessels and hemostasis in the event of impaired continuity of the blood vessel wall. The impaired hemo-static balance may lead on the one hand to an increased tendency to bleed, either spontaneously or only in response to an external stimulus. At the opposite end of bleeding are thrombophilic conditions characterized by an increased tendency to blood coagulation and thereby to the development of venous or arterial thrombosis. The hemostatic balance is the result of normal functioning of the blood vessel wall, platelets and plasma agents which include the coagulation and fibrinolytic systems and their inhibitors. By coagulation we understand the process leading to the formation of fibrin networks including the controlled interaction of coagulation factors. It is a physiological process as opposed to thrombosis which can be defined as increased coagulation under pathological conditions. Key words: coagulation - coagulation factors - coagulopathy - hemophilia - inhibitors of coagulation factors deficiencies - purpura - thrombocytopathy - thrombocytopenia.

Background Outcomes of total knee replacement in cases of hemophilic patients are worse than in patients who undergo operations due to osteoarthritis. Previous publications have reported varying rates of complications in hemophilic patients, such as infection and an unsatisfactory range of motion, which have influenced the survival of prostheses. Our retrospective study evaluated the data of hemophilic patients regarding changes in the development of the range of motion. Methods The data and clinical outcomes of 72 total knee replacements in 45 patients with hemophilia types A and B were reviewed retrospectively. Patients were operated between 1998 and 2013. All of the patients were systematically followed up to record the range of motion and other parameters before and after surgery. Results The mean preoperative flexion contracture was 17° ± 11° (range, 0°-40°), and it was 7° ± 12° (range, 0°-60°) postoperatively. The mean flexion of the knee was 73° ± 30° (range, 5°-135°) before the operation and 80° ± 19° (range, 30°-110°) at the last follow-up. The mean range of motion was 56° ± 34° (range, 0°-130°) before the operation and 73° ± 24° (range, 10°-110°) at the last follow-up. Conclusions Statistical analysis suggested that the range of motion could be improved until the 9th postoperative week. The patient should be operated on until the flexion contracture reaches 22° to obtain a contracture

Owing to the heterogeneity in the clinical phenotype of haemophilia A and B, it is now recognized that disease severity (based on factor VIII/IX activity) may no longer be the most appropriate guide for treatment and that a 'one-size-fits-all' approach is unlikely to achieve optimal therapy. Based on the present literature and consensus views of a group of experts in the field, this article highlights key gaps in the understanding of the diverse relationships between bleeding phenotype and factors such as joint health, genetic susceptibility, laboratory parameters, quality of life and management of pain. Early prophylaxis is a potential 'gold standard' therapy and issues surrounding inhibitor development, variations in its clinical use and long-term outcomes are discussed. Comprehensive treatment should be individualized for all patients (including those with mild or moderate haemophilia and carriers). Wherever possible all patients should be given prophylaxis. However, adult patients with a milder haemophilia phenotype may be candidates for ceasing prophylaxis and switching to on-demand treatment. Regardless, all treatment (on-demand and prophylaxis) should be tailored towards both the patient's personal needs and their clinical profile. In addition, as the associations between risk factors (psychosocial, condition-related and treatment-related) and clinical features are unique to each patient, an individualized approach is required to enable patients to alter their behaviour in response to them. The practical methodologies needed to reach this goal of individualized haemophilia care, and the health economic implications of this strategy, are ongoing topics for discussion.

Introduction Congenital dysfibrinogenemia and hypofibrinogenemia are rare diseases characterized by inherited abnormality in the fibrinogen molecule, resulting in functional defects (dysfibrinogenemia) or low fibrinogen plasma levels (hypofibrinogenemia). Materials and Methods We have described two abnormal fibrinogens – fibrinogen Hranice (γ Phe204Val) and Praha IV (γ Ser313Gly). The carrier of the Hranice mutation was a 40-year-old female with low fibrinogen levels. The carrier of the Praha IV mutation was a 42-year-old man with a history of idiopathic thrombosis, low functional fibrinogen levels, and a prolonged thrombin time. Results Fibrin polymerization kinetics measurement was normal in both cases (after the addition of either thrombin or reptilase), as well as was fibrinolysis. Scanning electron microscopy and confocal microscopy revealed significantly wider fibers in both cases, when compared with fibers prepared from healthy control samples. Although both cases are situated in the γ-nodule, they manifested differently. While the γ Ser313Gly mutation manifested as dysfibrinogenemia with a thrombotic background, the γ Phe204Val mutation manifested as hypofibrinogenemia without clinical symptoms. The mutation sites of both fibrinogens are in highly conserved regions of the fibrinogen γ chains. γ Ser313 is situated in a class 16:18 β hairpin and is involved in hydrogen bonding with γ Asp320. γ Phe204 is situated in an inverse γ turn and may be involved in π-π interactions. Conclusions Both mutations cause conformational changes in fibrinogen, which lead either to impaired fibrinogen assembly (fibrinogen Hranice) or abnormal fibrinogen function (fibrinogen Praha IV).

Objective. To evaluate the course of pregnancy and puerperium in asymptomatic carriers of FV Leiden and FII prothrombin mutation in heterozygous configuration in terms of risk of thrombembolic disease (TED) and late pregnancy complications. To evaluate whether global prophylactic LMWH administration during pregnancy benefits these women. Methods. We monitored the incidence of thrombembolic events and severe late pregnancy complications in 473 asymptomatic carriers of FV Leiden and FII prothrombin mutation in heterozygous configuration. In 253 women, preventive LMWH application was introduced already during pregnancy. In 220 women, the application of LMWH was commenced as late as on the delivery day. In both groups application of LMWH continued during the puerperium. Results. The incidence of TED in the whole group of carriers of thrombophylic mutations accounted for 0.19%. The incidence of severe late pregnancy complications was low - 2.5% compared with general population of pregnant women (6.4%). Conclusions. No direct causal relationship was established between asymptomatic carriage of Leiden and prothrombin mutation in heterozygous configuration and the occurrence of severe late pregnancy complications. There was no benefit from general LMWH prophylaxis started as early as pregnancy in these women and thus we consider it unnecessary.

Several studies suggest that recombinant activated factor VII (rFVIIa) is more cost-effective than plasma-derived activated prothrombin complex concentrate (pd-aPCC) in haemophilia with inhibitors. However, most do not consider differences between treated patients. This study compared the pharmacoeconomics of rFVIIa versus pd-aPCC treatment of mild to moderate bleeds in inhibitor patients, taking co-variables into account. Methods The HemoRec and HemIS registries capture exhaustive bleeding data in inhibitor patients in the Czech Republic. For each bleed, patient and bleed characteristics, treatment outcomes and bypassing agent use were retrospectively analysed, and direct costs of care per bleed calculated. Generalised Linear Model regression methods with cluster effect were employed to account for the possibility of several bleedings from the same patient. Results There were 108 and 53 mild to moderate bleeds in the rFVIIa and pd-aPCC groups, respectively. Although re-bleeding rates were similar in both groups, deeper analyses revealed significant differences in time to bleed resolution: 93.8% of bleeds treated with rFVIIa were resolved within less than 12h, versus 60.4% with pd-aPCC (P

Fibrinogen is a 340 kDA glycoprotein that plays a vital role in the hemostasis. The three pairs of polypeptide chains (Aα, Bβ and γ) form a heterodimer structure of fibrinogen, each chain is encoded by a distinct gene (FGA, FGB and FGG). Congenital dysfibrinogenemia is a rare qualitative disorder characterized by structural alterations in fibrinogen molecule resulting in its defective function. The clinical manifestation of majority cases is asymptomatic (50%); about 30% are associated with bleeding tendencies and only some cases are thrombotic (20%). In this study, we evaluated 25 patients with heterozygous missense mutations in exon 2 of FGA gene from 13 unrelated families, all with low levels of functional fibrinogen and prolonged clotting time. All mutations were located around thrombin cleavage site at N-terminus of a fibrinogen Aα chain. Mutation Aα G13E was found in 6 families (13 patients), Aα R16H in 4 families (6 patients), Aα R16C in 1 family (4 patients), and Aα R19G in 1 family (2 patients). Correlations between the genotype and phenotype were studied by fibrin polymerization, fibrinolysis, kinetics of fibrinopeptides release, and electron and confocal microscopy. The viscoelastic properties of fibrin clots were measured. All mutations caused a delay or absence of fibrinopeptide A release and prolonged fibrin polymerization. Clinical phenotypes of mutations were, unexpectedly and importantly, highly heterogeneous. Out of 25 patients 17 were asymptomatic, 7 had the bleeding tendencies and 1 patient had thrombosis. One woman with Aα G13E had 2 spontaneous abortions and 3 pregnant women with Aα G13E, Aα R16C and Aα R19G mutations suffered for hemorrhages or postpartum bleeding. Eventually, all had successful delivery under provided care with a hematologist. Small surgeries were performed to patients with Aα R16H supplied with fibrinogen concentrates and antifibrinolytics. Our observations show that the patients should be carefully treated based both on the results of the laboratory analyzes and personal and family history. This work was supported by the project of the Ministry of Health, Czech Republic, for conceptual development of research organization 00023736, by Grant from the Academy of Sciences, Czech Republic nr. P205/12/G118, and by ERDF OPPK CZ.2.16/3.1.00/24001. Disclosures No relevant conflicts of interest to declare.

Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Congenital hypofibrinogenemia is a rare autosomal bleeding disorder, either recessive or dominant, characterized by a low fibrinogen plasma level. A 28-year-old asymptomatic woman (fibrinogen Rokycany) and a 54-year-old man with thrombosis and pulmonary embolism (fibrinogen Znojmo) were investigated for a suspected fibrinogen mutation after abnormal coagulation tests results were obtained. DNA sequencing showed the heterozygous point mutation Bβ Asn351Lys in fibrinogen Rokycany and the heterozygous point mutation Bβ Arg237Ser in fibrinogen Znojmo, respectively. The kinetics of fibrinopeptide release was found to be normal in both cases. Fibrinolysis was impaired in the Znojmo variant. The average fibril diameters of Znojmo fibrin was slightly increased, but not differing significantly from normal; formed by less fibrils with abrupt fibril terminations. Rheological studies revealed a softer clot. Rokycany fibrin was formed by significantly narrower fibrils than normal fibrin; and the clot was denser than the control clot. Rheological studies revealed a stiffer clot. Impaired fibrinolysis and abnormal clot morphology may be the cause of thrombotic episodes in the patient with Znojmo mutation. New cases of hypofibrinogenemia and dysfibrinogenemia, found by routine coagulation testing, were genetically identified as a novel fibrinogen variants Bβ Asn351Lys (fibrinogen Rokycany) and Bβ Arg237Ser (fibrinogen Znojmo), respectively.

SummaryCongenital dysfibrinogenemia is a rare disease characterised by inherited abnormality in the fibrinogen molecule, resulting in functional defects. Two patients, a 26-year-old woman and a 61-year-old man, both with history of thrombotic events, had abnormal coagulation test results. DNA sequencing showed the heterozygous γY363N mutation (Fibrinogen Praha III) and the heterozygous Aα N106D mutation (Fibrinogen Plzeň), respectively. Fibrin polymerisation, after addition of either thrombin or reptilase, showed remarkably delayed polymerisation in both cases. Fibrinolysis experiments showed slower tPA initiated lysis of clots. SDS-PAGE did not show any difference between normal and Praha III and Plzeň fibrinogens. Both mutations had a significant effect on platelet aggregation. In the presence of either ADP or TRAP, both mutations caused the decrease of platelet aggregation. SEM revealed abnormal clot morphology, with a large number of free ends and narrower fibres of both fibrin Praha III and Plzeň. Praha III mutation was situated in the polymerisation pocket “a”. The replacement of the bulky aromatic side chain of tyrosine by the polar uncharged small side chain of asparagine may lead to a conformational change, possibly altering the conformation of the polymerisation pocket. The Plzeň mutation is situated in the coiled-coil connector and this replacement of polar uncharged asparagine residue by polar acidic aspartate changes the alpha-helical conformation of the coiled-coil connector;and may destabilise hydrogen bonds in its neighborhood. Although both mutations are situated in different regions of the molecule, both mutations have a very similar effect on fibrinogen functions and both are connected with thromboses.

Objective: A 22-yr-old woman had abnormal preoperative coagulation test results and congenital dysfibrinogenaemia was suspected. Patients and methods: The patient from Liberec (Czech Republic) had a low fibrinogen plasma level as determined by Clauss method, normal fibrinogen level as determined by immunoturbidimetrical method, and prolonged thrombin time. To identify the genetic mutation responsible for this dysfibrinogen, genomic DNA extracted from the blood was analysed. Fibrin polymerisation measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement and scanning electron microscopy were performed. Results: DNA sequencing showed the heterozygous fibrinogen γ Y262C mutation. Kinetics of fibrinopeptide release was normal, however fibrin polymerisation was impaired. Fibrinogen clottability measurement showed that only about 45% molecules of fibrinogen are involved in the clot formation. Scanning electron microscopy revealed thicker fibres, which were significantly different from the normal control. Conclusion: A case of dysfibrinogenaemia, found by routine coagulation testing, was genetically identified as a novel fibrinogen variant (γ Y262C) that has been named Liberec.

The transcription factor CCAAT/enhancer binding protein (C/EBP)alpha is a myeloid-specific transcription factor which is required for normal myeloid differentiation. C/EBPalpha is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. C/EBPalpha is a member of the basic region leucine zipper (bZIP) class of DNA-binding proteins. The loss of function of C/EBPalpha has leukemogenic potential. Four types of polymorphisms and 25 mutations (3 already known mutations and 22 novel mutations) were detected in CEBPA (gene for the transcription factor CCAAT/enhancer binding protein (C/EBP) alpha) in analysed samples from 390 patients with myelodysplastic syndrome (MDS) and hematologic malignancies. CEBPA mutations were found in 14/152 (9.2%) of acute myeloid leukemia (AML) patients' samples, 6/143 (4.2%) of MDS patients' samples, 2/56 (3.6%) of non-Hodgkin's lymphoma (NHL) patients' samples and 2/39 (5.1%) of multiple myeloma (MM) patients' samples. No C/EBPalpha mutations were detected in healthy donors (41 individuals). We discuss how these mutations can affect the cellular function of C/EBPalpha and block the myeloid differentiation.

Objectives: A 25-yr-old man from Prague had abnormal bleeding after several surgical operations with low fibrinogen level and hypofibrinogenemia was suspected. Patients and methods: The patient, 25 yr-old male had a low fibrinogen concentration as determined by the thrombin time and immunoturbidimetrical method. His 48-yr-old mother presented with normal coagulation tests, normal fibrinogen level and reported no history of bleeding. To identify the genetic mutation responsible for this hypofibrinogen, genomic DNA extracted from the blood was analyzed. Fibrin polymerization measurement, kinetics of fibrinopeptide release, fibrinogen clottability measurement, mass spectroscopy, and scanning electron microscopy were performed. Results: DNA sequencing showed heterogeneous fibrinogen γG351S mutation in the propositus. The mutant chain was found not to be expressed to the circulation by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Scanning electron micrographs of the patient's fibrin clot as well as kinetics of fibrinopeptide release and fibrin polymerization were found to be normal. Conclusion: A case of hypofibrinogenemia γG351S was found by routine coagulation testing and was genetically identified.

Introduction Various dysfibrinogenemias have been described worldwide. This paper describes two new cases of dysfibrinogenemia identified in the Czech Republic. Materials and methods The proposita of fibrinogen Nový Jicin, a 12-year-old girl, presented with hemorrhagic complications, low Clauss fibrinogen level (0.3 g/l) and prolonged both thrombin (70.8 s) and reptilase (> 180 s) time. Her mother and sister both presented with normal coagulation tests, normal fibrinogen level and reported no history of bleeding. The carriers of the fibrinogen Praha II were a 31-year-old man and his 11-year-old daughter. They both presented with low fibrinogen Clauss level (0.88 g/l) and prolonged thrombin and reptilase time. To identify the genetic mutation responsible for these dysfibrinogens, genomic DNA extracted from the blood was analyzed. The presence of the mutant chains in the circulation was determined by MALDI-TOF mass spectroscopy. Scanning electron micrographs of the patients' fibrin clots were obtained. Results The kinetics of fibrinopeptide release and fibrin polymerization were impaired for both fibrinogen Nový Jicin and Praha II. DNA sequencing showed heterogeneous fibrinogen Aα R16C mutation in the fibrinogen Nový Jicin case and heterogeneous fibrinogen Aα R16H in the fibrinogen Praha II case. The mutant chains were found to be expressed to the circulation by MALDI-TOF mass spectroscopy. Scanning electron micrographs of the patient's fibrin clot were found to be abnormal. Conclusions The case of dysfibrinogenemia Aα R16C-fibrinogen Nový Jicin and the case of dysfibrinogenemia Aα R16H were found by routine coagulation testing and were genetically identified.

Haemophilia registries, such as HemoRec, provide a valuable source of real-world data for the study of treatment modalities. We have reported that early treatment with rFVIIa appeared more effective than delayed-onset treatment in controlling the incidence of re-bleeding. The current study investigated whether time to treatment and/or initial dose of rFVIIa affects bleeding duration and, for the first time, their impact on acute pain control in inhibitor patients.

In the treatment of bleeds in haemophilia patients with inhibitors, a high initial dose of recombinant Factor VIIa (rFVIIa) provides at least equal efficacy and a similar safety profile to a standard initial dose. However, no pharmacoeconomic comparison between these dosing regimens has previously been performed. Here, we assess the pharmacoeconomics of high (120 μg/kg) versus standard (≤120 μg/kg) initial rFVIIa dose in inhibitor patients and the impact of time to treatment initiation on costs and outcomes.In a retrospective analysis, observational data on bleed characteristics, rFVIIa treatment, hospitalizations and outcomes were extracted from the Czech Republic HemoRec registry. Crude comparisons and generalized linear regression modelling (GLM; correcting for patient differences) were performed to compare costs and outcomes between the high and standard initial dosing groups.Of 314 rFVIIa-treated bleeding episodes (12 inhibitor patients), most were spontaneous joint bleeds and 67.5% were treated with a high initial dose. In the crude comparison, high initial rFVIIa dosing was associated with a lower mean number of doses needed to achieve haemostasis compared with standard dosing (p0.001), but higher total dose and costs (p ≤ 0.008). However, regression analyses revealed that high initial dose was associated with similar costs (p=0.891) and a shorter time to bleeding resolution (p=0.014). Increasing time to treatment initiation increased both time to bleeding resolution and total costs.Compared with a standard dose, a high initial rFVIIa dose may improve treatment outcomes without increasing costs. Early treatment initiation may reduce treatment costs.

Historically in hemophilia, outcome measures have not been collected systematically. Hence, there are insufficient clearly defined, evidence-based measures that can be applied consistently across hemophilia trials. This review focuses on some key challenges to evaluating patient outcomes and performing trials identified by experts at the Fourth and Fifth Zurich Haemophilia Forums. As procedures appear inconsistent across Europe, guidelines require modification to be more appropriate and/or realistically achievable. The outcome measures utilized, and the timing of their collection, should also be standardized, and more objective measures used where feasible. Implementation of outcome measures could be refined through greater understanding of patient heterogeneity, and tailored to differentiate between hemophilia- and aging-related disease effects. Furthermore, robust outcome measures that can also inform health-economic decisions are increasingly needed. Lastly, as patient recruitment poses a challenge, the panel proposed a call for action to motivate physicians and patients to participate in clinical trials. (Less)

Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aa, Bb, and g) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aa chain - fibrinopeptides A and the Bb chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined four unrelated children suspected dysfibrinogenemia. The first patient was a 5-yr old boy with prolonged thrombin time and low functional fibrinogen level. He presented with easy bruising and epistaxis. Genetic analysis revealed a heterozygous substitution Aalpha R16H. The second patient was a 13-yr old girl with prolonged thrombin and reptilase times and low functional fibrinogen level. She presented with menorrhagia. Genetic analysis revealed a heterozygous substitution Aalpha G17V. The third patient was a 12-yr old asymptomatic boy. Genetic analysis revealed a heterozygous substitution Aalpha R16H. Kinetics of fibrinopeptide release was impaired in all these cases. The fourth patient was a 3-yr old girl with low functional fibrinogen level. She presented with easy bruising. Genetic analysis revealed a heterozygous substitution Aalpha K448N. All patients presented with impaired fibrin polymerization. Conclusion All patients were found to be heterozygous for point mutations in FGA gene. Three mutations were found in the site of fibrinopeptide cleavage and one in the alphaC-domain. Mutations had different clinical manifestations from asymptomatic to bleeding. Mutations Aalpha Arg16His and Aalpha Gly17Val are among the most common fibrinogen mutations and both decelerate fibrinopeptide A cleavage from mutant fibrinogens. We describe here a novel previously unreported mutation Aalpha Lys448Asn affecting fibrin formation. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Disclosures No relevant conflicts of interest to declare.

Identifying haemophilia patients with inhibitors for clinical trials is difficult due to the limited number of patients available. Registries are therefore being established as an additional means of data collection. The aim of this study was to investigate the effect of different recombinant activated factor VII (rFVIIa; NovoSeven dose ranges and dosing schedules on the incidence of re-bleeding in haemophilia patients with inhibitors. In this retrospective, uncontrolled study, data on the bleeding patterns of adult haemophilia patients with high responding inhibitors were analysed. Only data from the Czech Republic, obtained by the HemoRec registry, were used. This study analysed 'real-life' clinical data and focused on the collection of the same parameters in different patients: time from bleeding onset to first injection, effect of first injection, number of re-bleedings, total number of injections and total amount of haemostatic drug used. Fifteen patients met the inclusion criteria and were included into the study (128 bleeding episodes). Patients treated within 2 h of bleeding onset experienced less re-bleeding than patients treated after 2 h of bleeding onset (5.2% vs. 13.7%, respectively). In addition, patients who were treated after 2 h of bleeding onset experienced fewer re-bleedings when high-dose rFVIIa was used (15.8% and 0%;120 microg kg(-1) and250 microg kg(-1), respectively). Initial high-dose rFVIIa was also associated with a decline in total rFVIIa consumption. This registry has provided a unique insight into the bleeding patterns of inhibitor patients, highlighting the importance of early treatment initiation and appropriate starting dose.

Development of inhibitors is a severe complication of haemophilia posing many management challenges. While a long-term goal in inhibitor patients is eradication of inhibitors through immune tolerance induction, bypassing agents such as recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (aPCC) are essential for control of bleeding episodes. Paediatric patients with haemophilia and inhibitors are at particular risk of recurrent haemarthroses, and management of these patients should seek to avoid joint damage and support the child's full social and physical development. Current options for management of bleeding complications include on-demand treatment of acute bleeding episodes, secondary prophylaxis to avoid recurrent bleeds and surgery to treat affected joints. There is also a rationale for adopting prophylactic approaches to prevent bleeding in inhibitor patients, allowing this group similar opportunities for protection against arthropathy development as are given to non-inhibitor patients. This paper, based on a roundtable meeting of haematology experts at the first Zurich Haemophilia Forum in May 2008, reviews the current evidence supporting more intense and prophylactic approaches to manage bleeding risk in paediatric haemophilia patients with inhibitors, and highlights the need for investigations of primary prophylaxis in this vulnerable patient group, to support best long-term outcome.

Abnormal coagulation properties indicative of a dysfibrinogen were found in the plasma of a 72-year-old male with multiple myeloma (IgGĸ, stage IIIA). The patient had high paraprotein concentration (85.75 g/l) and prolonged thrombin time (76.8 s), activated partial thromboplastin time (39.5 s), prothrombin time (23.5 s) and reptilase time (72.0 s). The fibrinogen level was increased. The fibrin polymerization induced by both thrombin and reptilase was impaired. Scanning electron microscopy revealed abnormal clot morphology. After six months of treatment, the paraprotein level decreased (19.48 g/l) and coagulation normalized as well as fibrin polymerization and fibrin clot morphology. It was found that the paraprotein interacts with the γ-chain of fibrinogen. Acquired dysfibrinogenemia associated with multiple myeloma was diagnosed in the 72-year-old patient.

Intrahepatic cholestasis of pregnancy (ICP) is a condition found in fewer than 1% of pregnant women in most countries. ICP typically develops in the second half of pregnancy as the result of defective bile acid transport, which leads to the release of liver enzymes and bile acids into the maternal circulation. Itching-the commonest symptom-may interfere with sleep, but in other respects the maternal disorder is benign and does not predispose to liver failure. The fetus, however, is at increased risk of preterm birth, intrauterine demise or stillbirth, and mecomium aspiration. This study compared two drugs, given separately and in combination. Both S-adenosyl-L-methionme (SAMe) and ursodeoxycholic acid (UDCA) enhance the excretory function of hepatocytes, stimulate choleretic activity, and dilute toxic bile acids in the enterohepatic circulation. UDCA also reduces the accumulation of bile acids in the fetus, and SAMe increases trans-sulfuration of bile acids, which increases their excretion. One or both of these drugs were given to 78 women with singleton pregnancies at less than 36 weeks' gestation who had moderate or severe ICP as evidenced by high self-reported symptom scores. One group of 25 patients received SAMe at a dose of 500 mg twice daily by slow running infusion for 12 days, followed by 500 mg daily by mouth until delivery. Another 26 women received UDCA orally in a dose of 250 mg 3 times a day. The remaining 27 women received both these treatments. The treatment groups were similar obstetrically at the outset. Although all 3 treatments resulted in improvement of maternal symptoms, more women had a favorable response to UDCA alone or in combination with SAMe than to SAMe alone. UDCA given alone or combined with SAMe resulted in greater decreases in the concentrations of bile acids and transaminases compared to SAMe alone, and a favorable response in most women (85% to UDCA alone and 89% to combined therapy). Monotherapy with SAMe did not significantly alter biochemical parameters. Only one patient, treated with UCA alone, became worse. The response was also more rapid-within 7 days of initiating treatment-with UDCA alone or in combination than in SAMe alone, but was fastest with combination therapy. However, by two weeks of therapy there was no difference between the two UDCA groups. There were no significant group differences in the duration of treatment, which ranged from 1 to 12 weeks. All treatments were well tolerated and no adverse side effects were recorded. No perinatal deaths occurred, but premature deliveries before 36 completed weeks of gestation exceeded the population average in all treatment groups. Birth weights below the 10th centile of national growth curves were, however, within the limits of the population average in all groups. UDCA, alone or combined with SAMe, appears to be an effective treatment for ICP. Antepartum and intrapartum monitoring of the fetus should be routine when severe ICP is present.

Introduction Hemostasis in childhood differs from that of adults; and the hemostatic system is still developing during childhood. These differences offer a protective advantage to children with hemorrhagic and thrombotic complications. Plasma levels of coagulation factors (except for fibrinogen, factor V and factor VIII), as well as plasma levels of protein C, protein S and antithrombin are reduced. Hereditary dysfibrinogenemia is a rare disorder wherein an inherited abnormality in fibrinogen structure may result in defective fibrin function and/or structure. Clinical symptoms may vary from asymptomatic to life-threatening bleeding complications. Venous or arterial thrombotic complications occur extremely rarely during childhood. Fibrinogen, a key component in hemostasis, is a 340-kDa glycoprotein. The molecule consists of three different pairs of polypeptide chains (Aα, Bβ, and γ) each encoded by a distinct gene (FGA, FGB, and FGG). N-terminal parts of the Aα chain - fibrinopeptides A and the Bβ chain - fibrinopeptides B are situated in the central part of the molecule and block polymerization of the molecules. Conversion of fibrinogen to fibrin occurs after the cleavage of N-terminal fibrinopeptides by the serine protease thrombin. Correct conformation of fibrinopeptides is important for the cleavage by thrombin. Methods Routine coagulation tests were performed with citrated plasma samples on a STA-R coagulation analyzer. The functional fibrinogen level was measured by the Clauss method. Total fibrinogen level was determined by an immunoturbidimetric assay performed on a UV-2401PC spectrophotometer. Fibrin polymerization induced by either thrombin or reptilase and fibrinolysis experiments were obtained by the turbidimetrical method. Fibrinopeptide release was measured as a function of time; and the fibrinopeptides were determined by the reversed-phase, high-performance liquid chromatography (RP-HPLC) method. The purified genomic DNA was amplified by polymerase chain reaction, using specific primers; and dideoxysequencing was performed with Dye Terminator Cycle Sequencing with a Quick Start kit and a CEQ 8000 genetic analysis system). Results We have examined two unrelated boys aged 4 (boy 1) and 14 (boy 2) for susp. dysfibrinogenemia. Routine coagulation tests revealed prolonged thrombin and reptilase time in both boys and also showed decreased functional fibrinogen levels in both kids (Tab. 1). Boy 1 presented with bleeding manifestation – easy bruising, epistaxis, and bleeding after tooth extraction. His 26-year-old mother presented with similar coagulation findings and with mild bleeding complications. Boy 2 was asymptomatic. Fibrin polymerization experiments carried out on plasma samples showed prolonged lag time and significantly reduced final turbidity in both cases. Measurement of kinetics of fibrinopeptide release showed a decreased amount of the released fibrinopeptide A in both patients. DNA sequencing of boy 1 revealed a point mutation in exon 2 of the FGA gene at the position 3456 G/A, which causes the substitution of Aα 16 Arg to His (fibrinogen Praha V). The boy was found to be heterozygous for the mutation as well as his mother. DNA analysis of boy 2 revealed the same point mutation in the FGAgene, causing the same substitution of Aα 16 Arg to His (fibrinogen Kralupy nad Vltavou). Conclusion In this study we report two cases of congenital defects in the fibrinogen Aα Arg16-Gly17 bond found during childhood. It has been described earlier that the replacement of Aα 16 arginine by histidine decelerates thrombin catalyzed fibrinopeptide A release. Although both kids presented with the same genetic defect and with similar coagulation results, they have different clinical manifestation of the disease. Acknowledgment This work was supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.16/3.1.00/28007. Table 1: Routine coagulation test results Boy 1 Boy 1's mother Boy 2 Normal APTT 40.1 s 34.6 s 35.3 s 27.5 - 36.1 s Prothrombin time 17.5 s 14.4 s 16.9 s 11.7 - 15.1 s Thrombin time 40.9 s 36.2 s 44.8 s 17.6 - 21.6 s Reptilase time 58.3 s 51.6 s 55.7 s 16.0 - 20.0 s Fibrinogen (Clauss) 0.51 g/l 1.12 g/l 0.58 g/l 2.00 - 4.20 g/l Fibrinogen (Immuno) 2.47 g/l 2.68 g/l 2.31 g/l 2.00 - 4.20 g/l Age 4 26 14 - Disclosures No relevant conflicts of interest to declare.

Three cases of abnormal fibrinogens: Sumperk (Bβ His67Leu), Uniaov (Bβ Gly414Ser), and Brno (γArg275His)

Abstract 3316 Objective: Recombinant FVIIa (rFVIIa) is indicated for the treatment of bleeding episodes in hemophilia patients with inhibitors at a dose of 90 mcg/kg every 2–3 hours, or as a single 270 mcg/kg rFVIIa dose (outside US). As patient response and bleeds are variable, treatment is often individualized by the center and by the patient/caregiver administering rFVIIa. With home treatment, it is difficult to ascertain how often patients use higher doses (>90 mcg/kg), and whether these doses are associated with increased risk of thromboembolic events (TEs). The aim of this analysis was to understand the safety of higher rFVIIa doses and the intervals to subsequent doses. Methods: Data for rFVIIa on-demand (OD) bleed treatment in inhibitor patients were obtained from 6 sources: 2 prospective randomized studies; 1 prospective observational US diary study (DOSE); the US-based Hemostasis and Thrombosis Research Society (HTRS) Registry; the international ONE Registry; and the HemoRec registry (Czech data only). Data on prophylactic (PPX) rFVIIa use was obtained from 3 sources: 1 prospective randomized trial; 1 retrospective chart review study (PRO-PACT); and the HemoRec registry (Czech data). Data on intervals between high initial/subsequent doses and any further bypassing agent dose in OD treatment were analyzed from 3 sources that reported time of administration (DOSE, ONE and HemoRec). All studies had data safety monitoring in the form of explicit surveillance, positive affirmations of lack of adverse events, or chart reviews. Overlap in patient participation in studies could not be ruled out; however, all data are believed to be non-overlapping due to study timelines. Demographics, rFVIIa dosing, and frequency of TEs were analyzed and reported using descriptive statistics. Results: A total of 481 inhibitor patients reported using 61,734 rFVIIa doses either for OD treatment or PPX. Most patients had hemophilia A (87%) and were Caucasian (79%). Age ranged from 18y). All data sources included the use of rFVIIa doses higher than current US-recommendations. Overall, 52.2% of doses (45.9% in adults) were >120 mcg/kg, the highest dose referenced from previous clinical trials in the US package insert. For the152 patients from DOSE, ONE and HemoRec included in the dosing interval analyses, age ranged from 18y). Patients received 3,042 rFVIIa doses for 1,017 bleeds. These included 361 initial rFVIIa doses (36%) >240 mcg/kg, including 136 followed by a second dose of bypassing agent (130 with rFVIIa; 15 with pd-aPCC). There were 670 doses >240 mcg/kg used throughout treatments (22%), including 327 followed by a subsequent dose (311 with rFVIIa; 23 with pd-aPCC). Most subsequent doses (79%) occurred after >8 hrs. Conclusions: A review of 61,734 cumulative rFVIIa doses in studies with explicit safety monitoring demonstrates the use and safety of rFVIIa doses >90 mcg/kg in inhibitor patients. When doses >240 mcg/kg were followed by other bypassing agent doses, the dosing interval was >8 hrs after 79% of doses and >24 hrs after 53% of doses. Unlike MedWatch and passive surveillance where underreporting is common, we expect that the explicit surveillance in these studies would adequately identify TE events. Thus, the lack of TEs supports the overall low TE occurrence (0.28%) reported in the original registration trials in inhibitor patients using ≤90 mcg/kg. These data reinforce that the arterial thrombosis risk identified in meta-analyses outside of licensed indications (critical bleeds) are unique to those populations and do not apply to inhibitor patients. Furthermore, higher doses may provide an opportunity to improve bleed treatment with less frequent injections and decreased follow-up infusions. Disclosures: Shapiro: Novo Nordisk inc.: Consultancy, Research Funding, Speakers Bureau. Off Label Use: rFVIIa use in indicated population of congential hemophilia with inhibitors at doses that differ from those in the prescribing information. Neufeld:Novo Nordisk Inc.: Consultancy, Research Funding. Blanchette:Novo Nordisk: Consultancy. Chambost:Novo Nordisk: Consultancy. Salaj:Novo Nordisk: Consultancy. Gut:Novo Nordisk Inc.: Employment. Cooper:Novo Nordisk Inc.: Employment.

Introduction Thrombosis is a common pathology underlying ischemic heart disease, stroke, and venous thromboembolism. D-dimer is a global indicator of blood coagulation activation and fibrinolysis and, therefore, an indirect marker of thrombotic activity. D-dimers half-life is 48 hours. D-dimer determination is the standard procedure in the treatment of thrombosis. Serine protease thrombin plays pivotal roles in thrombosis and hemostasis, blood coagulation and platelet activation. Thrombin has a short half-life and naturally occurring inhibitors, such as antithrombin, rapidly bind thrombin, which makes the direct measurements of in vivo active thrombin difficult. Thrombin binds to fibrin where it is quite efficiently protected from inhibition by heparin-antithrombin but susceptible to inactivation by different antithrombin-independent inhibitors. There are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrinogen E region, and one high affinity thrombin non-substrate binding site on fibrinogen gamma' chains inside the D region (Meh DA, Siebenlist KR, Mosesson MW, J Biol Chem. 1996, 23121-5). We have shown that thrombin bound to fibrin promotes further fibrin growth in the presence of fibrinogen and absence of free thrombin in solution (Riedel T, Brynda E, Dyr JE, Houska, M, J. Biomed. Mater. Res. Part A 2009, 437-447). The aim of this project was to look for any thrombin activity on isolated D-dimers using several commercial D-dimer kits in groups of patients with elevated D-dimers. Methods Three D-dimers kits were used (ELISA kits CEA506Hu, USCN and D-Dimer (D2D), BioAssay™; and immunoturbidimetric assay D-Dimer KAI-090, K-ASSAY). To detect thrombin activity on captured D-dimers one chromogenic (S-2238, Chromogenix) and two fluorogenic (SN-59, Haematologic Technologies, Inc.; (p-tosyl-Gly-Pro-Arg)2-R110, Thermo Fisher Scientific, Inc.) specific substrates and specific inhibitors (hirudine and PPACK) were used. Independently, bound proteins were removed from immobilized antibodies in 8 M urea and after treatment with trypsin analyzed by mass spectrometry (TripleTOF 5600, Sciex). 34 patients with high, moderate, and low levels of D-dimers and with different diagnosis were analyzed. Results Out of 18 patients with main diagnosis linked with thrombosis 61 % exhibited active thrombin on D-dimers. In these patients we found active thrombin bound to D-dimers captured by antibodies in all applied D-dimer kits. Using mass spectrometry thrombin bound to isolated D-dimers was detected. Specificity of thrombin activity related to D-dimers was determined by combination of several specific thrombin substrates and inhibitors. The activity of bound thrombin was remarkably stable over the period of more than 24 hours. It showed that precise measurement of even very low activity of thrombin was possible. In the group of 16 non-thrombotic patients with elevated D-dimers only 19 % exhibited thrombin activity. Interestingly, differences in values of thrombin activity were up to five orders of magnitude and differences in the activities related to the value of captured D-dimers were even greater. Conclusion This, to our knowledge, is the first report showing the presence of active thrombin bound to circulating D-dimers. Although the group of patients we were so far able to evaluate is too small to being statistically tested, the results are highly encouraging. The amount of bound active thrombin on fibrin degradation products reflects the way of thrombus formation and its degradation (times, durations and rates of relevant reactions and especially the rate of thrombin generation). The thrombin concentration present at the time of fibrin gelation plays an important role in formation of fibrin clot structure, including its mechanical and fibrinolytic stability. It remains to be seen in further studies with much larger cohorts of patients if the presence of active thrombin has any impact on pathophysiology of thrombosis and if its determination may be of importance for the improvement of diagnosis of thrombotic events. Supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.1.16/3.1.00/28007. Disclosures No relevant conflicts of interest to declare.

The tissue factor (TF) is one of the most important regulators of arterial thrombosis. Because arterial thrombosis is the pathophysiologic background of acute coronary syndrome, the possible impact of blocking the arterial thrombosis on its onset is a challenging problem. The investigations of TF brought a new concept of "cell-based coagulation model" which highlighted the question of blood-borne TF as a source of TF in circulating blood. In this review we summarize essential information on the pathophysiology, molecular structure, expression and distribution of TF and we propose a novel concept of blood-borne TF, suggesting the possibilities of inhibition of the coagulation cascade with newly synthetized drugs.