Severe Bleeding Tendency Research Articles

Coagulation Functions with Clinical Phenotype and Inhibitory Mechanisms in Acquired Factor V Inhibitors.

Abstract 2232Development of acquired factor V (aFV) inhibitors rarely occurs, but its clinical phenotype varies from asymptomatic to life-threatening bleeding. A recent systematic report describes that little bleeding symptom is present in 20% at the diagnosis for patients with acquired FV (aFV) inhibitors. However, the coagulation function and its mechanism(s) on the different clinical phenotype are poorly understood. In this study, we examined the coagulation function on aFV inhibitors by using comprehensive coagulation assays, thrombin generation test (TGT) and clot waveform analysis (CWA). TGT was performed using tissue factor (0.5 pM), phospholipid (PL; 4 μM) and ellagic acid (0.3 μM). CWA, that evaluates the parameters of min1 as maximum coagulation velocity and min2 as maximum coagulation acceleration, was performed on MDA-II® system. We tested 7 cases with aFV inhibitors. Four cases were asymptomatic (FV:C; 3.6±3.4 IU/dl, inhibitor 5.8±3.3 BU/ml: non-B group), and 3 cases had severe bleeding tendency (2.9 ± 4.5 IU/dl, 66 ±51 BU/ml; B group). In TGT, all cases in both groups little showed the thrombin generation within 60 min, independently of FV:C level and clinical phenotype, showing little informative in functional evaluation for aFV inhibitors. However, in a PT-based CWA, the clotting time observed in non-B group was markedly shorted compared to that in B group (62.2±17.0/112±15 sec; p=0.006). In addition, both parameters in non-B group were significantly greater than those in B group ( min1 ; 2.89±1.10/0.98±0.29 dT/dt; p=0.014) and ( min2 ; 0.75±0.40/0.15±0.07 d2T/dt2; p=0.028), suggesting that CWA was useful for the prediction and monitoring of hemorrhagic symptoms in patients with aFV inhibitors. To confirm the distinct mechanism(s) on both groups, the IgGs from aFV plasmas were immune-purified using protein G-Sepharose. In the reactant mixtures with normal plasma and aFV IgGs, all parameters obtained in CWA were similar to those obtained in patients’ plasmas. SDS-PAGE and western blotting revealed that 2 cases in B group reacted the light chain of FV(a). However, 2 cases in non-B group reacted the heavy chain, and other 2 cases were reacted with both chains (heavy>light), indicative of the distinct epitopes of IgGs in both groups. Since the light chain contains the PL-binding site(s), the effects of aFV IgGs were examined on the FV-PL binding in an ELISA. All IgGs in B group inhibited this binding (by 40–90%) dose-dependently, whilst little affected in non-B group. Since FV acts as a cofactor of activated protein C (APC) on inactivation of FVIIIa, the effects of aFV IgGs on the ability of APC on FVIIIa inactivation were examined using intrinsic FXa generation assay. The APC sensitivity ratio (APCsr) was expressed as ratio of amounts of generated FXa in the absence of APC relative to its presence. A low level of APCsr indicates the reduction in FVIIIa inactivation, consequently APC resistance. APCsr values in B group were >2.0 within normal range, whilst those in non-B group were decreased to 1.5, supportive of APCR in non-B group. Based on these findings, we propose that severe bleeding tendency in B group would be appeared through negligible prothrombinase activity, since aFV IgGs blocked the FV(a)-PL binding. While, clinical phenotype in non-B group would be asymptomatic, since aFV IgGs unaffect the FV(a)-PL binding and further cause the APC resistance. In addition, various clinical phenotypes in aFV inhibitors appear to be dependent on the recognizing epitope of these IgGs. Disclosures:No relevant conflicts of interest to declare.

The Cytokine Polymorphisms Affect the Severity of Thrombocytopenia at Diagnosis in Chronic Immune Thrombocytopenia.

AbstractAbstract 2194 Introduction:The severity of immune thrombocytopenia (ITP) depends on the degree of the thrombocytopenia and the extent of bleeding. Some investigators have reported the association between the thrombocytopenia and cytokine dysregulation in ITP. We investigated the association between the severity of thrombocytopenia at diagnosis in ITP patients and several cytokine polymorphisms, including IL-10-1082A/G, -819T/C, -592A/C, IL-17F-7488T/C and IL-18-607A/C, −137G/C. Patients and methods:We examined 102 patients (male/female, 24/78; median age, 42) diagnosed with chronic ITP. The definition, response criteria, including complete response (CR)and response (R), loss of CR,and “corticosteroid-dependence” were assessed according to the criteria of the ITP International Working Group. ITP with severe thrombocytopenia (ST group)was defined as thrombocytopenia (platelet count < 10×109/L) at the initial diagnosis of ITP. Genotyping of IL-10 (rs1800870 − 1082 A/G, rs1800871 − 819 T/C, and rs1800872 − 592 A/C) and IL-17F (rs763780, 7488 T/C) polymorphisms were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the genotyping of the IL-18 polymorphism (rs187238 −137G/C and rs1946518−607 A/C) was determined by the allelic specific polymerase chain reaction technique. To confirm the accuracy of the assay, amplification products of several individuals were sequenced using an ABI Prism Genetic Analyzer. Genotype and allele frequencies were compared between the study groups using χ2-test. The characteristics and laboratory features of ITP patients with each polymorphisms were compared using χ2-tests and student t-tests. Odds ratios (OR) and 95% confidence intervals (CIs) were estimated for each study. All patients were provided written information about the study. This study was approved by the Institutional Research Board of Gunma University Hospital.Results: Clinical features of chronic ITP:The platelet count ranged from 1×109/L to 98×109/L with a mean of platelet count of 32×109/L at the initial diagnosis. Fifty seven patients (49%) had bleeding tendency. Steroid treatment was given to 68 patients (66.7%) and eradication of Helicobacter pylori (H. pylori) was performed in 32 patients (31.4%), while splenectomy was performed in only 11 patients (10.8%). Clinical features of ST group vs. non-ST group in chronic ITP:Of these 102 patients, 17 (16.7%) had severe thrombocytopenia (platelet count < 10×109/L) (ST group). ST group were significantly older (ST group: median 59 years vs. non-ST group: 41 years, p<0.01) and had more severe bleeding tendency (ST group: 100% vs. non-ST group: 54%, p<0.0001). Steroid treatment was frequently given to ST group than to non-ST group (ST group: 100% vs. non-ST group: 59.5%, p<0.001). Though the response to corticosteroids treatment was not significantly different between ST group and non-ST group (CR rate, ST group: 50% vs. non-ST group: 51.0%, p=0.94), corticosteroid-dependent patients in ST group was significantly higher than in non-ST group (76.9% vs. 25.3%, p<0.005). Polymorphism study of ST group vs. non-ST group in chronic ITP:The frequencies of genotypes of cytokines in patients with chronic ITP according to the definition of criteria of ST were as follows: AA (93.3% vs. 97.1%) and AG (6.7% vs. 2.9%, p=0.48) for IL-10–1082; TT (46.7% vs. 33.3%), TC (33.3% vs.55 %) and CC (20% vs. 11.7%) for IL-10–819; AA (46.7% vs. 33.3%), AC (33.3% vs.55 %) and CC (12.2% vs. 11.5%) for IL-10–592; TT (100% vs. 81%) and TC (0% vs. 19%) for IL-17F; GG (82.4% vs. 74.4%), GC (17.6% vs. 23.2%) and CC (0% vs. 2.4%) for IL-18–137; AA (35.3% vs. 34.1%), AC (58.8% vs. 53.7%) and CC (5.9% vs 12.2%) for IL-18–607 loci (ST group vs. non-ST group, respectively). No significant difference was observed between ST group and non-ST group according to IL-10–1082A/G, −819T/C, −592A/C, and IL-18–607A/C, −137G/C polymorphism. However, the numbers of IL-17F 7488TT genotype (higher function type) in ST group were significantly higher than in non-ST group (ST group: 100% vs. non-ST group: 81% p<0.05). Conclusion:These findings suggest that severe thrombocytopenia at diagnosis have an impact of bleeding tendency and corticosteroid-dependency of chronic ITP. Furthermore, IL-17F polymorphism may affect the severity of thrombocytopenia of chronic ITP. Disclosures:No relevant conflicts of interest to declare.

Severe bleeding tendency caused by a rare complication of excessive fibrinolysis with disseminated intravascular coagulation in a 51-year-old Japanese man with prostate cancer: a case report

IntroductionDisseminated intravascular coagulation causes thrombotic tendency leading to multiple organ failure and occurs in a wide variety of diseases including malignancy. Disseminated intravascular coagulation is a latent complication in people with prostate cancer.Case presentationA 51-year-old Japanese man with advanced castration-resistant prostate cancer was admitted to our hospital because of extensive purpura and severe anemia. Prolonged plasma coagulation time, hypofibrinogenemia and normal platelet count suggested that a decrease in fibrinogen induced a bleeding tendency causing purpura. However, elevated plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers, with positive fibrin monomer test, manifested disseminated intravascular coagulation and subsequent fibrinolysis. Plasma levels of thrombin-antithrombin complex, fibrin and/or fibrinogen degradation products and D-dimers decreased after administration of low-molecular-weight heparin. However, low fibrinogen and α2-antiplasmin levels were not improved and plasmin-antiplasmin complex did not decrease, which revealed excessive fibrinolysis complicated with disseminated intravascular coagulation. We suspected that prostate cancer cell-derived urokinase-type plasminogen activator caused excessive fibrinolysis. Administration of tranexamic acid for fibrinogenolysis was added together with high-dose anti-androgen therapy (fosfestrol) for prostate cancer. Thereafter, prostate-specific antigen and plasmin-antiplasmin complex decreased, followed by normalized fibrinogen and α2-antiplasmin levels, and the patient eventually recovered from the bleeding tendency. Immunohistochemical staining of the biopsied prostate tissue exhibited that the prostate cancer cells produced tissue factor, the coagulation initiator, and urokinase-type plasminogen activator.ConclusionThis patient with rare complications of disseminated intravascular coagulation and excessive fibrinolysis is a warning case of potential coagulation disorder onset in patients with prostate cancer. We propose that combined administration of tranexamic acid and low-molecular-weight heparin together with high-dose anti-androgen therapy is a useful therapeutic option for patients with this complicated coagulation disorder.

Spontane Hämatomneigung und Hüftschmerzen bei einer 65-jährigen Patientin

The case of a 65-year-old woman with acquired hemophilia is reported. Acquired hemophilia is characterized by the development of inhibitors directed against coagulation factors. Impairment of plasmatic hemostasis leads to a severe bleeding tendency in individuals without a preexisting coagulation defect with considerable mortality. Pathophysiology, diagnostic work-up, and treatment are summarized and discussed.

Retinoids in Pediatric Onco-Hematology: the Model of Acute Promyelocytic Leukemia and Neuroblastoma

Retinoids are lipophilic compounds derived from vitamin A, which have been extensively studied in cancer prevention and therapy. In pediatric oncology, they are successfully used for the treatment of acute promyelocytic leukemia (APL) and high-risk neuroblastoma (HR-NBL). APL is a subtype of acute myeloid leukemia (AML) clinically characterized by a severe bleeding tendency with a highrisk of fatal hemorrhage. The molecular hallmark of this disease is the presence of the promyelocytic leukemia (PML)-retinoic acid receptor-α (RAR α) gene fusion that plays a critical role in promyelocytic leukemogenesis and represents the target of retinoid therapy. The introduction in the late 1980s of all-trans retinoic acid (ATRA) into the therapy of APL radically changed the management and the outcome of this disease. Presently, the standard front-line therapeutic approach for pediatric APL includes anthracycline-based chemotherapy and ATRA, leading to a complete remission in almost 90% of the patients. Neuroblastoma (NBL) is an aggressive childhood tumor derived from the peripheral neural crest. More than half of patients have a high-risk disease, with a poor outcome despite intensive multimodal treatment. Although the exact mechanism of action remains unclear, the introduction of 13-cis-retinoic acid (13-cis-RA) in the therapy of NBL has improved the prognosis of this disease. Currently, the standard treatment for HR-NBL consists of myeloablative therapy followed by autologous hematopoietic stem cell transplantation (HSCT) and maintenance with 13-cis-RA for the treatment of minimal residual disease, leading to a 3-year disease-free survival rate (DFS) of about 50%. In this paper the authors provide a review of the peer-reviewed literature on the role of retinoids in the treatment of pediatric APL and HR-NBL, summarizing the most relevant clinical trial results of the last decades, analyzing the ongoing trials, and investigating future therapeutic perspectives of children affected by these diseases.

Case report: use of all-trans retinoic acid in pregnancy

Acute promyelocytic leukemia (APML) is a rare disease, associated with abnormal promyelocytes in bone marrow and a severe bleeding tendency. The disease should be managed as a medical emergency, and...

A case of acquired FXIII deficiency with severe bleeding symptoms

Acquired factor XIII (FXIII) deficiency due to an autoantibody against FXIII is a very rare, yet potentially life-threatening bleeding disorder. As the standard coagulation tests (prothrombin time and activated partial thromboplastin time) are normal, the specialized tests are required to make an accurate diagnosis. Here, we report a case of acquired FXIII deficiency with severe bleeding symptoms. A 75-year-old man was referred to our hospital because of severe bleeding tendency after a tooth extraction. Laboratory findings showed that routine coagulation studies were normal, but factor XIII (FXIII) activity was low (3%). The presence of FXIII inhibitor was detected with dot blotting studies. Although the bleeding tendency was very severe, it was successfully controlled by infusion of FXIII concentrates combined with immunosuppressive treatment (oral prednisolone). Fibrin cross-linking study showed the significant delay of the γ-chain dimer and α-chain polymer formation. Western blotting revealed the marked decrease in FXIII-A level. The mixing study of FXIII activity measured using amine-incorporation assay showed the incomplete inhibition pattern. There seems to be little agreement as to the treatment strategy of acquired FXIII deficiency. In this patient, the use of FXIII concentrates was very useful in the initial treatment of bleeding symptom. The use of steroids was also effective in increasing FXIII activity without any serious complications.

Platelet Dysfunction in a Patient with Multiple Myeloma: A Case Report with a Literature Review

Multiple myeloma is a monoclonal plasma cell proliferation disorder with various symptoms and signs caused by paraproteinemias. Among these signs, a bleeding tendency is one of the major fatal causes. However, significant severe bleeding is rare in most cases. In this study, we report a case of multiple myeloma in a patient who had a severe recurrent bleeding tendency due to platelet dysfunction caused by paraproteins. After being treated with therapeutic plasma exchange and chemotherapy, the patient`s monoclonal protein level decreased and the bleeding stopped. (Korean J Med 2012;83:823-827)

Management of the Sponataneous Bleeding Episodes in Factor VII Deficiency. A Prospective Evaluation of the STER,

Abstract 3368 IntroductionPatients with an inherited factor VII (FVII) deficiency may display a wide range of clinical phenotypes, from an asymptomatic condition to serious hemorrhagic episodes such as fatal central nervous system (CNS) or gastrointestinal (GI) bleeds (Mariani G et al. Thromb Haemost 2005; 93: 481–7). Symptomatic patients can be divided into two major categories: those with mild-to-moderate bleeding tendency and individuals with a severe bleeding tendency which may be more severe than that in hemophilia. The former group mainly experience mucosal bleeding, a clinical picture that mimics that of a platelet disorder and often does not call for treatment. In contrast, for the most severely affected patients, characterized by life- or limb-threatening hemorrhages, more aggressive Replacement Therapy (RT) as long or short-term prophylxis may be required. AimWithin the frame of the Seven Treatment Evaluation Registry we have prospectively collected large numbers of treatments for spontaneous bleeding episodes and evaluted schedules and side-effects. Clinicaltrials.gov identifier: NCT01269138. ResultsAs of May 2011, 64 evaluable spontaneous bleedings (managed in 16 haemophilia treatment centres, 11 countries), were observed in 40 FVII-deficient subjects (25 females and 15 males) with residual FVII coagulant activity (FVIIc) levels between <1% and 20% (90% of patients had FVIIc below 5% of normal). Bleeding episodes were grouped into the following categories: 1. CNS + GI (n=2+4), 2. Hemarthroses (n=23), 3. Muscle hematomas (n=6), Epistaxis (n=4), Gum bleeding (n=7), Menorrhagia (n=11), Other (n=7) (hemorrhoidal n=2, subcutaneous hematomas n=2, easy bruising n=1, hematuria n=1, hemoperitoneum n=1). Recombinant FVIIa was given to treat 47 episodes, plasma derived FVII (pdFVII) concentrates 7, Prothrombin Complex Concentrate (PCC) 3 and Fresh Frozen Plasma (FFP) 7 (Table 1). No thrombosis episodes were reported during the thirty-day follow up.Table 1Type of Bleeding, Replacement Therapy (RT) schedules and outcomeBleedsnRTRT daysn. of dosesTotal dose range (μg/Kg IU/Kg ml/Kg)OutcomeCNS & GI6rFVIIa 31–41–8rFVIIa 160-240median 160Excellent 1 P.Effective 2pdFVII 2pdFVII 15-60–P.Effective 1 Effective 1FFP 1FFP 2–Effective 1Hemarthrosis23rFVIIa 221–21–7rFVIIa 18 – 110060Excellent 19 Effective 3PCC 1PCC 38–Excellent 1Hematomas (muscle & subcut.)8rFVIIa 41–61–12rFVIIa 54- 8066Excellent 3 Effective 1pdFVII 1pdFVII 56–Excellent 1PCC 2PCC 30–Excellent 1 n.e. 1FFP 1FFP 72–Effective 1Menorrhagia11rFVII 101–61–8rFVII 20 – 12041Excellent 5 Effective 5pdFVII 1pdFVII 135Effective 1Epistaxis & Gum bleeding12rFVIIa 511–3rFVIIa 27- 18071Excellent 4 Effective 1pdFVII 4pdFVII 21-5644Excellent 3 Effective 1FFP 3FFP 1.8-122.6Effective 3Other5rFVIIa 31-111-52rFVIIa 33-1716†33Excellent 1 Effective 2pdFVII 1pdFVII 18–Excellent 1FFP 1FFP 31–P.Effective 1*patient treated for severe hemoperitoneum; P.Effective:Partly Effective; n.e.: not evaluable ConclusionTreatment schedules and outcomes depend on the clinical setting. As for the severest bleeds (CNS and GI), on demand RT is probably not the best choice and prophylaxis should be considered as first line option especially in babies and children. Hemarthroses can be successfully treated for one day only; the number of doses used vary (from 1 to 7) as variable is the total dose of rFVIIa (from 35 to 210 μg/Kg/bw, mean 73.7, median 60) but an average intermediate dose of 60 μg/Kg/bw, possibly split in two doses, seems appropriate. The same holds for muscle and subcutaneous hematomas, always with one-day treatment schedule. RT protocols for menorrhagia varied either in terms of duration or rFVIIa total dose (1 to 6 days, total rFVIIa dosing ranging from 20 to 135 μg/Kg/bw, mean 88, median 40); treatment duration, however, did not appear to influence the outcome as one day RT showed the same efficacy as multiple days schedules. Successful RT for gum bleeding and epistaxis required treatment for one day only with doses of rFVIIa ranging from 27 to 180 μg/Kg/bw (mean 87.8, median 71) and of pdFVII ranging from 21 to 56 IU/Kg/bw (mean 41.5, median 44.5). Others bleed management were reported (hemorrhoidal, hematuria, easy bruising, peritoneal bleeding) but numbers do not allow to draw any conclusion as is the case for treatments carried out with FFP and PCCs. Disclosures:Knudsen:Novonordisk: Employment.

International Consortium for the Study of Clinical and Molecular Aspects of Bernard-Soulier Syndrome

Abstract 707▪FN2▪This icon denotes a clinically relevant abstractBernard-Soulier syndrome (BSS) is an extremely rare inherited bleeding disorder characterized by a defect of the GPIb/IX/V complex, which is essential for hemostasis, as the GPIbα subunit binds to subendothelial von Willebrand factor. Since the identification of the first mutation in 1990, almost one hundred cases carrying mutations in the GP1BA, GP1BB, and GP9 genes have been described. Most of the mutations prevent the coordinated association of the complex or binding to the von Willebrand factor. BSS is usually transmitted as a recessive trait with giant platelets and severe bleeding tendency. However, there are families with a dominant mild form, in which the affected individuals have only moderate macrothrombocytopenia and bleeding tendency.A correct definition of the clinical and laboratory features, together with accurate genotype/ phenotype correlation studies, remains essential for understanding the molecular basis of the disease and managing patients appropriately. Moreover, it is important to understand the variability of clinical manifestations. Since BSS is rare with an estimated prevalence of 1:1,000,000, an International Consortium has recently been established to collect a large series of cases and families worldwide.At present, the Consortium has been compiling data from 165 unrelated families, of which 50% have not been previously described. In this cohort, the molecular genetic testing reveals more than 30 novel mutations, confirming the wide spectrum of alterations responsible for the disease. Data from 65 unrelated families (69 patients) mainly from France, Italy and Japan show that 23 have mutations in GP9 and 29 in GP1BB. In the remaining 13 families, the defective gene is GP1BA. In agreement with the view that BSS is an extremely rare disease, 53 probands carried homozygous mutations, 10 are compound heterozygous, and 2 hemizygous because of a 22q11 deletion of the DiGeorge syndrome.The mean age of patients at diagnosis was 18 years (range 0–75 years) of which 27 were males and 38 females. Misdiagnosis of autoimmune thrombocytopenia was frequent and 26 patients were previously treated with steroids, intravenous immunoglobulins and/or splenectomy. Except two Japanese cases without any bleeding manifestations, patients presented with a variable bleeding diathesis measured by the World health Organization bleeding scale: grades 1, 2, 3 and 4 in 9, 18, 19 and 10 patients, respectively.The mean platelet count was 64×109/L (range 24–130) as determined by microscopy. In contrast, using a cell counter, thrombocytopenia was more severe (45×109/L; range 5–125). The mean platelet mean diameter was larger than in controls and varied from 2.9 to 7.5 mm. Ristocetin-induced platelet agglutination was absent or lower than 22% of normal response in all patients.Flow cytometry revealed a defective expression of the GPIb/IX/V expression in all patients. Correlating between expression data and gene affected, we found that the expression of GP1ba was often undetectable in patients with GP1BA mutations whereas it was higher, 8% and 17%, in patients with mutations of GP1BB and GP9, respectively. Instead, the GPIX mean level was 14%, 8% and 25% in patients with GP9, GP1BB and GP1BA mutations. The expression of GPV was higher than that of the other subunits, being more than 30% regardless of which gene was mutated.This is the largest cohort of BSS patients characterized to date. These patients together with the other 100 cases not yet included in the BSS database will enable correlations of the molecular genetic defects, receptor expression and clinical manifestations observed in BSS patients. Disclosures:Zieger:CSL Behring Hattersheim: Research Funding.

A severe neonatal presentation of factor II deficiency

Prothrombin deficiency is an autosomal recessive disorder associated with moderate or severe bleeding tendency. In this study, a three-month-old boy with non-consanguineous parents was referred for convulsions because of intracerebral hemorrhage. Standard coagulation tests revealed that the patient's plasma prothrombin activity was 12%, while his father's and mother's levels were 55% and 70%, respectively. Analysis of the prothrombin gene revealed that this patient is a compound heterozygote for two missense mutations: one maternally inherited point mutation in the propeptide (p.Arg4Gln) and one paternally inherited mutation in the kringle-2 (p.Arg220Pro) domain. Structural analysis was performed and confirmed that the resulting mutations were inferred to respectively affect the cleavage of the propeptide from the Gla domain, and the stability of the kringle-2 domain, both resulting in a severe hypoprothrombinemia. In unusually bleeding newborn of non-consanguineous parents, rare severe homozygous bleeding disorders need to be considered to facilitate early diagnosis and treatment.

Identification and Cloning of Novel Mutations In a Compound Heterozygous Factor V Deficient Patient

AbstractAbstract 2210 Introduction:Congenital factor V (FV) deficiency is a rare clotting disorder associated with mild to severe hemorrhagic symptoms and a prevalence of approximately 1 per million in the general population. Patients normally present with very low or unmeasurable levels of functional and/or immunoreactive FV and are usually homozygous or compound heterozygous for mutations located in the F5 gene. Heterozygotes typically have approximately half-normal levels of FV and are asymptomatic, whilst compound heterozygotes may show mild to severe bleeding diathesis. Patient Description:A proband now aged 73, was clinically diagnosed since childhood as having severe FV deficiency (<3% FV activity by clinical lab analysis), exhibiting severe bleeding tendency with surgery or dental extraction. Other symptoms included frequent nose bleeds, bruising easily and profound menorrhagia that led to eventual hysterectomy in mid-thirties. Additional routine clinical assays for clotting factors were normal. There was no history of parental bleeding tendency. Methods:DNA was isolated from peripheral blood leucocytes. Exonic and flanking intronic sequences of F5 were amplified by PCR and subjected to automated nucleotide sequencing. To investigate the role of identified mutations in FV function and protein secretion, wildtype (WT) FV and FV variants consistent with mutations identified for the patient using the QuikChange Site-Directed Mutagenesis Kit were cloned into the pED-FV-1033 expression vector. This produced recombinant single chain FV that requires proteolytic activation to express cofactor function. Plasmids encoding WT FV, and the mutation identified in the patient were transfected into baby hamster kidney cells (BHK) using Lipofectamine and stable clones were established. Secretion of FV protein was quantified by commercial enzyme immunoassay (EIA), and FV activity was evaluated using conventional prothrombin time (PT) and activated partial thromboplastin time (APTT) clotting assays. Western blot analysis using monoclonal antibodies to both FV heavy (FVaH) and light chains (FVaL) detected FV antigen and fragmentation profiles. Results:Based on the lack of parental bleeding, compound heterozygosity was probable for this FV deficient patient because the DNA sequence analysis revealed two novel mutations; the first changed the codon for Leu1821 to Ser (L1821S), the second changed Gly2192 to Cys (G2192C). Clotting assays showed low patient plasma FV activity of 0.5±0.015% for PT and 2.0±0.10% for APTT compared to normal pooled plasma. Western blot analysis demonstrated the patient FV banding pattern was comparable to normal plasma, whilst densitometric analysis of the specific bands showed that the patient had 9% of the normal FV antigen level. Recombinant FV secretion detected by EIA from various clones ranged from 0 to 16.6ng/mL. L1821S (28%) and G2192C (25%) mutants secreted lower FV protein compared to WT. Clotting assays (n=2) showed that Leu1821S activity levels were 15% that of WT in contrast to G2192C where no FV clotting activity was detectable. Compared to WT, L1821S had 55% and G2192C had only 1.4% of the specific activity.Western blot analysis demonstrated the L1821S banding pattern was comparable to WT whilst G2192C lacked several fragments. Prolonged thrombin (FIIa) pretreatment of FV clones confirmed WT and both mutants were cleaved to FVaL (∼74k Da) and FVaH (∼105 kDa). In addition, FIIa pre-treatment enhanced clotting activity for most of the clones evaluated. Interestingly, the inactive G2192C mutant acquired modest FV function after pre-incubation with FIIa. Conclusions:Two novel FV mutations that affect both function and secretion have been identified in a FV deficient patient and have been cloned. The L1821S mutation is located close to a FV metal ion binding site that may affect function. G2192C introduces a thiol into FV, reported in the literature to have deleterious effects on protein folding, which may explain attenuated secretion and lack of clotting activity. Only after prolonged pre-treatment with FIIa, G2192C function was acquired, which may suggest that this mutation stabilizes the pro-cofactor conformation of FV. Low in vitro secretion of mutant FV compared to WT corresponds to low patient plasma FV antigen, which is compounded by profound loss of function in G2192C. Further biochemical studies are underway to understand the effect of G2192C on FV activity. Disclosures:No relevant conflicts of interest to declare.

Successful treatment of disseminated mixed invasive fungal infection after hematopoietic stem cell transplantation for severe aplastic anemia

Concomitant infections are frequent and usually the causes of death in patients with severe AA. HSCT can restore hematopoiesis in AA, but it is usually life threatening when patients simultaneously have an IFI. Mixed IFIs have been reported on rare occasions. The exact diagnosis of IFIs is difficult because of low fungus culture rate, difficultly obtaining tissue specimens in severely immunocompromised patients or those with bleeding tendencies. Otherwise, treatment with anti-fungal drugs alone for DMIFI was always lethal in previous reports. Surgical resection is crucial for invasive zygomycosis, but severe pancytopenia and bleeding tendency make therapy difficult. Herein, we report that with a combination of aggressive anti-fungal drugs, HSCT, and surgery, we successfully treated a 10-yr-old boy with severe AA and pulmonary zygomycosis before HSCT and disseminated mixed invasive zygomycosis and aspergillosis after HSCT.

Thrombin generation in patients with factor XI deficiency and clinical bleeding risk

Factor XI (FXI) deficiency is a rare bleeding disorder. Most patients with FXI deficiency are mild bleeders but certain patients with similar FXI activity exhibit different bleeding phenotype. Routine laboratory assays do not help physicians to estimate the individual bleeding risk in these patients. Thrombin generation test (TGT) is a more comprehensive, global function test of the clotting system. We investigated whether or not the bleeding tendency of patients with FXI deficiency is correlated with features of the TGT. Twenty-four patients with FXI deficiency were divided in two groups: (i) severe bleeders (n = 9) and (ii) mild or non-bleeders (n = 15). All severe bleeders had a personal history of surgery-related severe bleeding. Thrombin generation (TG) was measured in platelet-rich plasma (PRP) using a low concentration of tissue factor 0.5 pm. In patients exhibiting severe bleeding tendency, independently of their FXI level, a dramatically impaired TG was observed. For example, despite a low plasma FXI = 1 IU dl(-1), a clinically non-bleeding individual exhibited normal TG results whereas another patient with severe bleeding history and FXI = 40 IU dl(-1) had a very low TG capacity. Low velocity and delayed TG were the main parameters suggesting a higher bleeding risk. DNA analysis of patients reported eight novel mutations of the FXI gene but neither mutation location nor secretion or not of the variant correlated with the bleeding tendency. The results of this study suggest that TG measurement in PRP may be a useful tool to predict bleeding risk in FXI deficiency and should be studied further in larger prospective clinical studies.

Angeborene Thrombozytenfunktionsstörungen

Inherited thrombocytopathies are much less frequent in comparison to acquired platelet function disorders. However, congenital disorders can lead to severe bleeding tendency and are often not diagnosed. They are induced by different platelet defects based on disorders of platelet adhesion, receptors, secretion and signal transduction. In some cases they are associated with thrombocytopenia, giant platelets and various comorbidities. This article gives an overview regarding diverse defects, their diagnosis and treatment options.

Gene analysis and prenatal diagnosis for two families of congenital factor V deficiency

We studied two families in which the probands had severe bleeding tendency and showed low plasma levels of coagulation factor V (FV) antigen and activity. Sequence analysis of the FV gene on proband 1 demonstrated a novel G16088C homozygous missense mutation in exon 3 resulting in an Asp 68 to His substitution and on proband 2, a C69969T homozygous missense mutation in exon 23 leading to Gly2079Val. The parents of both families were each heterozygous for the corresponding FV gene defect. During their second pregnancy, the two families requested prenatal diagnosis. Chorionic villi were analysed at 12 weeks of gestation and cord blood samples were tested at 22 weeks. Microsatellite analysis performed in family 1 showed that the foetus sample was not contaminated by maternal tissue. The foetus 1 was found to be heterozygous for the familiar G16088C mutation with lower FV activity in the cord blood; the foetus 2 was a normal one. The diagnosis was confirmed after the birth. This is the first report of prenatal diagnosis for FV deficiency.

Impact of Age at Diagnosis on Clinical Features in Children with Anomalous Arrangement of the Pancreaticobiliary Duct

In patients with an anomalous arrangement of the pancreaticobiliary duct (AAPBD), clinical presentations may differ between infants and older children. The optimal timing of surgery remains controversial, particularly in early infancy. The aim of this study was to evaluate the clinicopathological features and clinical outcomes using comparative methods between infants and older cases. MATERIALS AD METHODS: From 1983 to 2007, a total of 85 consecutive children with AAPBD were treated at our institute. They included 46 with the cystic type, 33 with the fusiform type, and 6 with the non-dilatation type. These patients were divided into 2 age groups: "infant" (n=9), <12 months old; and "older", >1 year old (n=76). A retrospective study was performed. Mean age was 5.2 months (range, 8 days-11 months) in the infant group and 5.2 years (range, 1.2-17.3 years) in the older group. Jaundice was significantly more frequent in the infant group ( P<0.05), whereas abdominal pain was more common in the older group ( P<0.001). Bleeding tendencies such as cranial hemorrhage or bloody stools were noted in only 3 infants. In terms of liver histology, liver cirrhosis was observed in 2 infants, one of whom was a 3-month-old girl with severe jaundice resulting in living-donor liver transplantation, despite bile drainage. A single postoperative death occurred due to an adenocarcinoma arising in a choledochal cyst in a 12-year-old girl. Problems characteristic of infantile AAPBD were a severe bleeding tendency and irreversible liver cirrhosis, which could develop as young as 3 months old. The surgical recommendation for infantile AAPBD is thus early surgery before the age of 3 months to prevent liver failure.

Factor VIII and von Willebrand factor – too sweet for their own good

Although factor VIII (FVIII) and von Willebrand factor (VWF) are products of two distinct genes, they circulate in plasma as a tight non-covalent complex. Moreover, they both play a critical role in the haemostatic process, a fact that is illustrated by the severe bleeding tendency associated with the functional absence of either protein. FVIII is an essential cofactor for coagulation factor IX, while VWF is pertinent to the recruitment of platelets to the injured vessel wall under conditions of rapid flow. FVIII and VWF have in common that they are heavily glycosylated: full-length FVIII contains 20 N-linked and at least seven O-linked glycans, while VWF contains 12 N-linked and 10 O-linked glycans. Three decades of research have revealed that the carbohydrate structures of FVIII and VWF contribute to many of the steps that can be distinguished in the life-cycle of these proteins, including biosynthesis/secretion, function and clearance. In this review, several of these aspects will be discussed. In addition, the interaction of the FVIII/VWF complex with two families of carbohydrate-binding proteins, i.e. Galectins and Siglecs, and their potential physiological relevance will be discussed.

Inherited Disorders of Platelet Function in Pediatric Clinical Practice: A Diagnostic Challenge

Hereditary disorders of platelet function are a heterogeneous group of diseases that are often complex and tend to go undetected until clinically relevant bleeding occurs. Hallmarks are epistaxis, easy bruising, mucous membrane bleeding, perioperative bleeding and menorrhagia. Bleeding may be intermittent and unpredictable. After decades of successful research on platelet biology and genetics, research findings have not been satisfactorily translated to clinical practice. The lack of robust and well- standardized test systems continues to make the diagnosis of platelet defects cumbersome for the practising clinician. Patient history and description of clinical bleeding symptoms are essential. Exclusion of von Willebrand disease, platelet count and investigation of blood smears may provide a tentative diagnosis. Light transmission aggregometry is still considered the gold standard for assessing platelet function. Due to the wide range of possible genetic defects molecular biological analyses can complement but do not substitute for other tests. The true incidence of inherited disorders of platelet function is unknown. A survey in Germany revealed that receptor-defects including Glanzmann's thrombasthenia and Bernard-Soulier syndrome and aspirin-like defects were the most frequently diagnosed platelet disorders. Of affected children 60% presented with mild and 40% with moderate to severe bleeding tendency. Epistaxis, cutaneous and mucous membrane bleeding were the most frequent symptoms. The paediatric competence network of the GTH e.V. comprises 44 collaborating centres that are caregivers to over 150 children with well-defined inherited platelet defects. A major goal of this network is to promote diagnosis of children with inherited disorders of platelet function.

Severe Elbow Arthropathy in a Patient with Congenital Afibrinogenemia

Afibrinogenemia is a rare coagulation deficiency, inherited as an autosomal recessive trait. It can be manifested in homozygotes or compound heterozygotes by a severe bleeding tendency caused by deficiency of fibrinogen (factor I). The estimated prevalence in Italy is about 1:1,000,000, and 0.2% of the patients with a coagulation deficiency in the United Kingdom have afibrinogenemia1,2. In Switzerland, nine patients are listed in the national hemophilia registry. Among severe bleeding symptoms, umbilical cord bleeding is the most frequent and life-endangering event3. Intracranial hemorrhage is the major cause of death and is the primary reason for prophylaxis. Less severe findings, such as spontaneous muscle hematomas, epistaxis, and menorrhagia, are also frequent3. Patients with afibrinogenemia have remarkably fewer spontaneous hemarthroses in contrast to better known bleeding disorders such as hemophilia A and hemophilia B3-6. We report the case of a patient with congenital afibrinogenemia who had severe elbow arthropathy and was treated with a total elbow arthroplasty. The patient was informed that data concerning the case would be submitted for publication, and he consented. A forty-seven-year-old man was referred to our institution with a severe arthropathy of the left, nondominant elbow. Afibrinogenemia was diagnosed at birth. For many years, he was administered 4 g of Haemocomplettan HS (human fibrinogen; CSL Behring, Bern, Switzerland) intravenously every two weeks. The causative mutation in his family (the parents were nonconsanguineous, and his brother and two cousins were also affected) was identified as a homozygous deletion of approximately 11 kb of the fibrinogen alpha chain gene7. Over the years, the left elbow became increasingly symptomatic secondary to recurrent hemarthroses. The elbow was always the primary joint involved, although mild osteoarthritic changes were noted in the ankles. In 1991, at the age of …