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Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n = 50), systemic lupus erythematosus (SLE; n = 40), and the antiphospholipid antibody syndrome (APS; n = 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay.

Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n = 50), systemic lupus erythematosus (SLE; n = 40), and the antiphospholipid antibody syndrome (APS; n = 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay.

This study prospectively evaluated the rate of biologic response to desmopressin (DDAVP) in 66 patients with type 1 or 2 von Willebrand disease (VWD), each of whom had, on the basis of available records, a clinically significant bleeding history and at least one of the following laboratory abnormalities: bleeding time (BT) longer than 15 minutes, ristocetin cofactor activity (VWF:RCo) less than 10 IU/dL, factor VIII coagulant activity (FVIII:C) less than 20 IU/dL (severe VWD). Before the study, responsive patients were defined as those who, 2 hours after infusion of 0.3 μg/kg DDAVP, had increased baseline values of VWF:RCo and FVIII:C by at least 3-fold and achieved levels of at least 30 IU/dL for both and a BT of 12 minutes or less. The rate of biologic response varied according to VWD types and was higher in type 1 (7 of 26, 27%) than in type 2 (7 of 40, 18%) (type 2A [1 of 15, 7%], type 2M [3 of 21, 14%], type 2N [3 of 4, 75%]). Mutations in the VWF gene were previously known or newly identified in most patients with types 2A (n = 15 of 15), 2M (n = 15 of 21), and 2N (n = 4 of 4), but in none of those with type 1 VWD. Genotype provided more information than phenotype in predicting individual responses to DDAVP only in patients with 2A and 2N VWD. This prospective study showed that the rate of biologic response to DDAVP is relatively low not only in type 2 but also in type 1 VWD when uniform and stringent criteria for patient selection and responsiveness are applied.

This study prospectively evaluated the rate of biologic response to desmopressin (DDAVP) in 66 patients with type 1 or 2 von Willebrand disease (VWD), each of whom had, on the basis of available records, a clinically significant bleeding history and at least one of the following laboratory abnormalities: bleeding time (BT) longer than 15 minutes, ristocetin cofactor activity (VWF:RCo) less than 10 IU/dL, factor VIII coagulant activity (FVIII:C) less than 20 IU/dL (severe VWD). Before the study, responsive patients were defined as those who, 2 hours after infusion of 0.3 μg/kg DDAVP, had increased baseline values of VWF:RCo and FVIII:C by at least 3-fold and achieved levels of at least 30 IU/dL for both and a BT of 12 minutes or less. The rate of biologic response varied according to VWD types and was higher in type 1 (7 of 26, 27%) than in type 2 (7 of 40, 18%) (type 2A [1 of 15, 7%], type 2M [3 of 21, 14%], type 2N [3 of 4, 75%]). Mutations in the VWFgene were previously known or newly identified in most patients with types 2A (n = 15 of 15), 2M (n = 15 of 21), and 2N (n = 4 of 4), but in none of those with type 1 VWD. Genotype provided more information than phenotype in predicting individual responses to DDAVP only in patients with 2A and 2N VWD. This prospective study showed that the rate of biologic response to DDAVP is relatively low not only in type 2 but also in type 1 VWD when uniform and stringent criteria for patient selection and responsiveness are applied.

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P?

Elevation of serum lipoprotein (a) (Lp[a]) is a known risk factor predisposing to cardiovascular and cerebrovascular disease. However, little is known about the role of increased Lp(a) in venous thromboembolism (VTE). This study evaluated the role of Lp(a) among a panel of established hereditary thrombogenic defects in patients with VTE. A total of 685 consecutive patients with at least one episode of VTE and 266 sex- and age-matched healthy controls were screened with regard to activated protein C resistance, protein C, protein S, and antithrombin deficiency, elevated serum levels of Lp(a), and the factor V G1691A, MTHFR C677T, and prothrombin G20210A mutations. Elevated Lp(a) levels above 30 mg/dL were found in 20% of all patients, as compared to 7% among healthy controls (P< .001, odds ratio [OR] 3.2, 95% confidence interval [CI], 1.9-5.3). The coexistence of FV G1691A and elevated Lp(a) was significantly more prevalent among patients with VTE than in the control group (7% versus 0.8%; P< .001, OR 9.8, 95% CI, 2.4-40.7). No other established prothrombotic risk factor was found to be significantly combined with increased Lp(a). These data suggest that Lp(a) concentrations greater than 30 mg/dL are a frequent and independent risk factor for VTE. Furthermore, elevated Lp(a) levels might contribute to the penetrance of thromboembolic disease in subjects being affected by other prothrombotic defects, such as FV G1691A mutation.

Approximately 25% of hemophilia A patients infused with factor VIII (fVIII) mount an immune response, which leads to its inactivation. Anti-fVIII autoantibodies are also seen rarely in individuals with normal fVIII. We have previously demonstrated that some anti-A2 and anti-C2 domain antibodies are fVIII inhibitors and that many patients have additional inhibitors with a fVIII light chain (LCh) epitope outside C2. Because the contribution of the different antibodies to the plasma inhibitor titer had been examined in a limited number of patients (14), we report in this study a more extensive analysis of 55 plasmas. The dominant inhibitors in 62% (13 of 21) of autoantibody plasmas were directed only against C2 or A2, but not both, whereas this pattern was found in only 15% (5 of 34) of hemophilic plasmas. In addition, anti-A2 inhibitors were present in 71% (24 of 34) of hemophilic plasmas, but only 33% (7 of 21) of autoantibody plasmas. These results demonstrated that the inhibitor response in hemophiliacs was more complex and the epitope specificity was somewhat different. A comparison of hemophiliacs treated only with plasma fVIII or recombinant fVIII showed no significant differences in the complexity of the inhibitor response, as = 2 different inhibitor antibodies were present in 78% (18 of 23) of the former and 82% (9 of 11) of the latter. In contrast, the major inhibitors in 35% (8 of 23) of hemophiliacs treated with plasma fVIII were directed against C2 and another LCh epitope within residues 1649-2137, but not A2, while none (0 of 11) treated with recombinant fVIII had this pattern.

No relevant conflicts of interest to declare.

Radsak: Celgene: Research Funding.

Von Willebrand factor (VWF) is secreted as an acute phase protein during inflammation. The main mechanism regulating the size and prothrombotic activity of VWF is the specific proteolytic activity of ADAMTS-13. To determine the relevance of this regulatory pathway for the innate inflammatory response by polymorphonuclear neutrophils (PMN), we employed a mouse model of invasive pulmonary aspergillosis (IPA) where PMN functionality is crucial for fungal clearance and survival. IPA was induced by intratracheal application of Aspergillus fumigatusconidia in wild-type (129/Sv/Pas) or Adamts13 deficient (Adamts13-/-) mice. After PMN depletion using a anti-Gr-1 specific antibody, all mice infected with Aspergillus fumigatusconidia developed neutropenia and succumbed due to lethal IPA. In contrast, all undepleted wild-type mice survived the infection. Interestingly, Aspergillus fumigatusinfection in Adamts13-/-mice was lethal in 20% of the animals displaying a more severe course of IPA, as indicated by an increased fungal burden in lung homogenates along with increased levels of albumin and the inflammatory mediators IL-1β, IL-6, TNF-α, KC and MCP-1 in the bronchio-alveolar lavage fluid (BALF) compared to wild-type controls. Beyond this, we observed a decreased number of PMN in BALF of infected Adamts13-/-mice compared to wild-type mice. Lung histology sections demonstrated a more pronounced perivascular leukocyte infiltration in further support of a dysregulated inflammatory response in Adamts13-/-mice. Importantly, we observed no general defect in the activation of neutrophil effector functions as demonstrated by the normal induction of the oxidative burst, phagocytosis, degranulation, L-selectin shedding and apoptosis in response to formyl-peptide receptor agonists or exposure to Aspergillus fumigatusconidia or hyphae in vitro. Therefore, we conclude that the proteolytic regulation of VWF by ADAMTS-13 in an important mechanism to control PMN recruitment in the regulation of the innate inflammatory response in invasive fungal infections.

With marketing authorization in 2005, a non-interventional study (SET = Surveillance of Efficacy and Tolerability) with a double virus inactivated VWF/FVIII concentrate (Wilate®) was initiated in Germany. In 2012, the inclusion of patient documentation was terminated for final evaluation of the study data, representing the treatment of 170 patients suffering from von Willebrand’s disease (VWD).The presented study was performed to assess the haemostatic efficacy and safety of a newly introduced VWF/FVIII product in the treatment of all types of VWD patients in every day clinical setting and to validate the results from pivotal clinical trials.Patients of any age suffering from hereditary or acquired VWD, requiring replacement therapy were included. Apart from demographic and anamnestic data, details of all injections for treatment of bleeding episodes, surgeries and prophylactic treatment were documented and assessed. Clinical efficacy and tolerability were rated by the treating physicians using four-point verbal scales. All data underwent a pre-defined data management process including double data entry and plausibility checks by an independent statistical institute.Data of 170 patients suffering from VWD and representing the broad spectrum of disease severity was provided by 26 treatment centers. About two thirds of the patients are female; with an age range from 1 to 85 years at study entry. Six cases of acquired VWD and 7 type 3 VWD patients were included. Type 2 VWD of various subtypes accounts for about 30% of the patients with the remaining patients suffering from type 1 VWD. 108 surgical procedures in 82 patients were documented. In all evaluated surgical procedures, the efficacy of the concentrate was rated to be “excellent” or “good”. Efficacy in 156 bleeding episodes was assessed as “excellent/good” in 95.2% of ratings. 13 patients underwent a prophylactic treatment regimen which resulted in a drop of bleeding frequency to below 1 bleeding episode per month. Five patients had experienced suspected adverse drug reactions - all without sequelae. The ADR rate per injection was as low as 0.1%.The results presented reflect the experience of routine use of Wilate® in all types of VWD in various clinical settings. They confirm the excellent efficacy and tolerability which had been demonstrated previously during an extensive panel of clinical trials.Alesci: Octapharma AG: Investigator Other. Von Depka:Octapharma AG: Investigator Other, Speakers Bureau. Feddern:Octapharma AG: Employment. Halimeh: Octapharma AG: Investigator Other, Research Funding. Hegener: Octapharma AG: Employment. Kadar:Octapharma AG: Investigator Other. Miesbach: Octapharma AG: Investigator Other. Nowak-Göttl:Octapharma AG: Investigator Other, Research Funding. Scharrer:Octapharma AG: Investigator Other.

With marketing authorization in 2005, a non-interventional study (SET = Surveillance of Efficacy and Tolerability) with a double virus inactivated VWF/FVIII concentrate (Wilate®) was initiated in Germany. In 2012, the inclusion of patient documentation was terminated for final evaluation of the study data, representing the treatment of 170 patients suffering from von Willebrand's disease (VWD).

Thirteen children up to 12 years of age and 14 patients being 65 or older were treated for prophylaxis, haemorrhages or peri-operatively. Obvious deviations were found in general condition, bleeding locations and types of surgeries for the elderly patients. Further, the data provide evidence of age group-related differences regarding reason for administration (see table 1) and dosages administered. The efficacy and tolerability of wilate®treatment in children and elderly was as high as in the total group.Young or old age seem to have an impact on clinical requirements of VWD. However, larger cohorts are required to confirm these first findings of the non-interventional study “Wilate SET”.Hegener: Octapharma AG: Employment. Feddern:Octapharma: Employment.

Abstract 4630

No relevant conflicts of interest to declare.

Abstract 2251

Thrombotic thrombocytopenic purpura (TTP) is characterized by microthrombi, hemolytic anemia as well as thrombocytopenia. These symptoms are caused by a decreased activity of the protease ADAMTS13 which cleaves the von Willebrand Factor (VWF), due to mutation of the ADAMTS13-gene or autoantibodies. At the moment, the only available immediate therapy is plasmapheresis with Fresh Frozen Plasma (FFP) which may induce side effects. Therefore an alternative therapy might be the treatment with clotting factor concentrates.40 plasma samples were tested, consisting of FFP and solvent/detergent treated plasma, four batches of each blood group; VWF/VIII concentrates (Wilate®; Wilfactin®; Haemate®P; Immunate®; Kogenate®, Beriate®). In all samples ADAMTS13 activity, antigen and autoantibodies against ADAMTS13 were investigated. Additionally, the samples were tested for the presence of ultralarge VWF multimers. The BCS method according to Böhm, two ELISAs (Technozym®ADAMTS13 and Actifluor™ADAMTS13) and the electrophoresis on a SDS gel were used.ADAMTS13 activity was found in all VWF)VIII concentrates, which are produced from human plasma, but only with a very low activity (Wilate® 5.6%; Wilfactin® 2.8%; Haemate®P 13%; Immunate® 3.7%). ADAMTS13 activity was not detectable in the factor VIII concentrates (Kogenate® <2%; Beriate® <2%). Usual FFP and solvent/detergent treated plasma samples, contained much higher ADAMTS13 activity and antigen values than concentrates (FFP 78.6%, solvent/detergent treated plasma 77.6%). However a difference of activity between individual blood groups was evident in FFP samples (blood group A 69.4%; B 64.9%; AB 98.1%; 0 82.0%). Ultralarge VWF multimers could be demonstrated in VWF containing concentrates, less in VIII concentrate from human plasma and not in FFP and solvent/detergent treated plasma samples. As expected recombinant-produced VIII concentrate contained no traces of ultralarge VWF. In all analyzed samples antibodies were negative.For therapy of TTP clotting factor concentrates cannot be used as an alternative to the usual FFP and solvent/detergent treated plasma, because their ADAMTS13 activity values are too low.In addition, the VWF/VIII concentrates contain higher quantities of ultralarge VWF multimers, which are contraindicated for TTP patients. The differences of ADAMTS13 activity in FFP samples varies between the individual blood groups and batches. Solvent/detergent treated plasma shows less variation in ADAMTS13 activity due to the manufacturing process involving plasma pooling.No relevant conflicts of interest to declare.

The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates.We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers.The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level.All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13.No relevant conflicts of interest to declare.

Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia, hemolytic anemia and microthrombi. A deficiency of the metalloprotease ADAMTS 13, which cleaves a Tys1605-Met1606 bond in the A2 subunit of von Willebrand factor (VWF), leads to formation of ultra large von Willebrand multimers (UL-VWF) and can cause platelet aggregation and mircovascular thrombosis. Treatment of choice is the substitution of plasma with plasmaexchange. There are two different plasma types available: Fresh Frozen Plasma (FFP) and solvent/detergent (s/d) treated plasma. This treatment may carry significant risks and side effects for the patients. Therefore we investigated the side effects of the therapy and furthermore the ADAMTS13 activity of the two plasma types.A questionnaire was send to 66 TTP patients of the Department of Hematology to evaluate different side effects of the therapy. 20 batches of FFP and 4 batches of s/d plasma of all blood groups were investigated on ADAMTS13 activity. The ADAMTS13 activity was detected with BCS-Method according to Böhm and two commercial FRET assays.So far 34 patients were inquired about age, weight and suspected trigger situations that might have caused their TTP manifestation. The mean age of the patients was 34 years with a mean weight of 70kg. A previous infection caused TTP manifestation in 42% of the patients; drug therapy (22%) and pregnancy (17%) were other mentioned triggers. 94% of the patients suffered from an acquired TTP and only 6% had a hereditary TTP. The patients had 2.88 relapses and were treated with 16.27 plasmaexchanges. 56% had an additional therapy with Rituximab to achieve a faster remission of the disease. These patients needed less plasmaexchanges for recovery, which proofed to be significant at 2% level in a one sided t-test. Tingling (64.7%) and shivering (51%) were the most often mentioned side effects and simultaneously described as the strongest. Shivering was significantly correlated to tachycardia (p<0.01). Headaches were significantly correlated to hot flushes, tingling and collapse (p< 0.05). Side effects and allergic reactions occurred in the therapy with FFP as well as with s/d plasma. Another side effect was the complication that came along with infection of the venous access. Most patients had a central venous catheter (72%) and described infections and pruritus (60%), 50% of them mentioned this complication more than once. We found in usual FFP slightly higher ADAMTS13 activity levels (696.97 ng/ml) than in s/d virus inactivated plasma (643.86 ng/ml). The ADAMTS13 activity varied between the different assays (normal range: 666 ± 135ng/ml).Our investigation demonstrated that plasmaexchange therapy is still associated with a wide range of side effects. Side effects of plasmaexchange that were most frequently described by patients were tingling and shivering. Headaches also occurred in various cases. Patients suffered generally from more than one side effect at the same time during the treatment. Allergic reactions to the plasma therapy were mentioned by 65% of the patients.No relevant conflicts of interest to declare.

Abstract 3678

Abstract 3677

Abstract 4661

A severe functional deficiency in the ADAMTS13 protease, characteristic for patients suffering from thrombotic thrombocytopenic purpura (TTP), results in persisting prothrombotic ultra-large von Willebrand factor (ULVWF) multimers, which can lead to systemic microvascular thrombosis. The congenital form of TTP is attributed to the presence of homozygous or double heterozygous mutations in the ADAMTS13 gene leading to impaired secretion and/or a functional defect of the protease. Decreased ADAMTS13 activity has been also described for several physiological and pathological conditions. Emerging evidence indicates mild ADAMTS13 deficiency might also increase risk for arterial thrombosis such as for ischemic stroke. We therefore investigated a possible causal relation between moderate ADAMTS13 deficiency and ischemic stroke in one patient selected with the lowest ADAMTS13 activity amongst a cohort of 70 patients with ischemic stroke. At age 34 and 46 years the patient had suffered from idiopathic ischemic strokes with negative common prothrombotic risk factors. We found slightly elevated VWF antigen (161%), ULVWF multimers and reduced ADAMTS13 activity (17%). Genetic analysis identified five nucleotide exchanges in the ADAMTS13 gene leading to one heterozygous missense mutation P353L and four homozygous amino acid polymorphisms (R7W, Q448E, P618A, A732V). Her asymptomatic son presented with normal VWF antigen (99%) and an ADAMTS13 activity of 58% transmitted by a mutant maternal allele and a paternal allele harboring the single Q448E polymorphism. Recombinant expression studies indicated P353L alone causes an impact on ADAMTS13 secretion that is further pronounced in the context of the four polymorphisms. Secreted ADAMTS13 containing P353L or P353L combined with the four polymorphisms presented with similar binding interaction in a binding assay with immobilized multimeric VWF, however, the binding affinity was slightly less than that of wild-type ADAMTS13 or polymorphic rADAMTS13 proteins containing either Q448E alone or all four polymorphisms combined. ADAMTS13 activity analysis suggested that mainly the secretion process or the protein stability rather than the proteolytic activity was affected by the mutations. Co-transfections mimicking the patient’s genotype indicated homozygosity of the four polymorphisms in the patient led to an attenuation of the adverse effect of the heterozygous P353L point mutation resulting in the moderate ADAMTS13 deficiency in the patient. Our results suggest moderate familial ADAMTS13 deficiency might be causally involved in the pathogenesis of ischemic stroke.

A severe functional deficiency in the ADAMTS13 protease, characteristic for patients suffering from thrombotic thrombocytopenic purpura (TTP), results in persisting prothrombotic ultra-large von Willebrand factor (ULVWF) multimers, which can lead to systemic microvascular thrombosis. The congenital form of TTP is attributed to the presence of homozygous or double heterozygous mutations in the ADAMTS13 gene leading to impaired secretion and/or a functional defect of the protease. Decreased ADAMTS13 activity has been also described for several physiological and pathological conditions. Emerging evidence indicates mild ADAMTS13 deficiency might also increase risk for arterial thrombosis such as for ischemic stroke. We therefore investigated a possible causal relation between moderate ADAMTS13 deficiency and ischemic stroke in one patient selected with the lowest ADAMTS13 activity amongst a cohort of 70 patients with ischemic stroke. At age 34 and 46 years the patient had suffered from idiopathic ischemic strokes with negative common prothrombotic risk factors. We found slightly elevated VWF antigen (161%), ULVWF multimers and reduced ADAMTS13 activity (17%). Genetic analysis identified five nucleotide exchanges in the ADAMTS13 gene leading to one heterozygous missense mutation P353L and four homozygous amino acid polymorphisms (R7W, Q448E, P618A, A732V). Her asymptomatic son presented with normal VWF antigen (99%) and an ADAMTS13 activity of 58% transmitted by a mutant maternal allele and a paternal allele harboring the single Q448E polymorphism. Recombinant expression studies indicated P353L alone causes an impact on ADAMTS13 secretion that is further pronounced in the context of the four polymorphisms. Secreted ADAMTS13 containing P353L or P353L combined with the four polymorphisms presented with similar binding interaction in a binding assay with immobilized multimeric VWF, however, the binding affinity was slightly less than that of wild-type ADAMTS13 or polymorphic rADAMTS13 proteins containing either Q448E alone or all four polymorphisms combined. ADAMTS13 activity analysis suggested that mainly the secretion process or the protein stability rather than the proteolytic activity was affected by the mutations. Co-transfections mimicking the patient's genotype indicated homozygosity of the four polymorphisms in the patient led to an attenuation of the adverse effect of the heterozygous P353L point mutation resulting in the moderate ADAMTS13 deficiency in the patient. Our results suggest moderate familial ADAMTS13 deficiency might be causally involved in the pathogenesis of ischemic stroke.

The acquired von Willebrand syndrome (AVWS) is a bleeding disorder, which is amongst others associated with myeloproliferative diseases. Its prevalence varies from 0,04–0,13% in literature. Typical laboratory findings such as a low VWF:RCo/VWF:Ag- Ratio, a prolonged aPTT, PFA and bleeding time, a decrease of VWF:RCo, FactorVIII:, VWF:Ag, typical VWF multimeric structure and the absence of a family history of bleeding are the basis for the diagnosis of AVWS. Bleeding episodes are typical for AVWS and are mostly of the mucocutaneous type (epistaxis, heavy menstrual bleeding, gingival and postoperative bleedings). Myeloproliferative diseases, particularily essential thrombocythemia, are with 15–18% the third most frequent observed comorbidity of patients with AVWS, but the prevalence of AVWS in patients with MPS is -at our knowledge- still unknown. We investigated 54 patients with the diagnosis of myeloproliferative disease (44 ET, 9 PV, 1 IMF), which was established according to the WHO-criteria over a period of 6 years (2000–2006). By analysing the typical laboratory parameters for AVWS, the VWF multimeric structure and the own and family history of bleeding we detected that 67% (36/45) of these patients had an AVWS: 27/44 ET (61%), 8/9 PV (89%), 1/1 IMF. The two most sensitive parameters in this context were VWF:RC/vWF:Ag- Ratio and VWF:RCo. It is well known that AVWS can disappear by treating the MPD. We could also show that treating the MPD with Hydroxycarbamid platelets, VWF multimeric structure, VWF:RCo/VWF:Ag-Ratio and VWF:RCo and the bleeding tendency were normalized. The JAK2-V617F mutation is frequently found in myeloproliferative disorders (MPD): Up to 97% patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) carry this mutation. A further goal in this investigation was to find out a correlation between JAK2-V617F mutation, MPD and AVWS. JAK2-V617F diagnosis was performed in 27 of the 54 patients with MPD. 10 of these 27 patients carried a mutated JAK2-allele. However we observed no significant difference between mutated and unmutated patients in correlation to AVWS. In our investigation the high prevalence of 67% AVWS in patients with MPD confirm the hypothesis that until now AVWS is an underdiagnosed disease. Because of this high prevalence, the possibility of regression by treating MPD and the importance of early diagnosis for prevention of unexpected, sometimes letal bleeding complications, it would be benefical to introduce the AVWS- diagnosis-procedure as a routine step for patients with MPD.

Plasma exchange (PE) is the treatment of choice in thrombotic thrombocytopenic purpura (TTP) patients. The use of PE has decreased the mortality rate of TTP from 90 to 10–30%. However PE is costly, technically demanding and often associated with serious complications such as infection and/or transfusions reactions. Some patients are also unresponsive to PE. In recent studies patients with refractory TTP have achieved sustained remission after Rituximab treatment. Patients with chronic TTP are very often afraid of PE. We cared for 4 anxious patients, who refused PE treatment on the one side and demanded treatment in their early phase of relapse on the other side. Therefore we treated 4 patients with a beginning relapse of TTP with Rituximab (375 mg/m2/week) before onset of symptoms. The 4 patients had already suffered from 10, 3, 4 and 5 relapses. Due to close control of laboratory data in an 8 or 12 weekly interval it was possible to detect deterioration of laboratory data (decrease of platelets <50.000, increase of LDH>500 U/l, decrease of ADAMTS-13 activity to <6.5%, increase of inhibitor to ADAMTS-13 >4BU and detection of schistocytes in the blood smear). Already after the first or second Rituximab dose a significant effect on the laboratory data was seen (increase of platelets >100.000, decrease of LDH to normal values, increase and normalization of ADAMTS-13 activity, decrease of inhibitor titer and disappearance of schistocytes). In the mean observation time (3–12 months) no deterioration of laboratory data and no TTP symptoms were observed. No side effects of Rituximab occurred. Based on our experience we recommend close control of laboratory parameters and an immediate treatment before onset of symptoms in patients with impending relapses. Up to now the reasons for these rapid effects of Rituximab are not fully understood. Other mechanisms of Rituximab treatment in TTP compared to lymphoma therapy might be possible.

Recent research efforts have provided significant insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). However, the low incidence of the disease hampers the identification of trigger factors initiating an acute TTP episode. In the present work data from 68 TTP patients were analyzed. The patients were asked by a questionnaire to provide data concerning age, gender, medical history, underlying and concomitant diseases and potential triggers. Additional laboratory and clinical data from onset of disease and remission phase were obtained by a second questionnaire completed by the physicians. Our study showed that long term abuse of nicotine is a predisposing factor with an OR of 3.19. The OR for obesity and development of TTP was 2.01. The influence of nicotine abuse has not yet been described whereas the association between obesity and development of TTP is already known. The intake of oral contraceptives (13 of 46 women/28%) of Tamoxifen, of Fluvastatin, of Ticlopidine and of Quinine was identified to be potential triggers. 55% (34 patients) of the patients reported infections and inflammations as triggers (mostly influenza (n=16) and gastroenteritis (n=14)). The relation of female and male patients was 3:1. The mean age, in which the initial TTP episode occurred, was significantly lower in female patients compared with the male patients (33.8 versus 43.8 years). Severe ADAMTS-13 deficiency was found in 91% in all investigated episodes of the patients. Inhibitory activity was associated with severe ADAMTS-13 deficiency in 81% of the cases. The comparison of patients during remission with and without relapses of TTP revealed a significant lower ADAMTS-13 activity in patients with relapsing TTP compared to patients without a relapse of TTP. This result suggests that ADAMTS-13 activity has prognostic value with respect to the occurrence of relapse.

Introduction. FXIII deficiency is a rare autosomal recessive disorder affecting approximatly 1 out of 1–3 million inhabitans. The disease is characterized by bleeding, impaired wound repair and spontaneous abortions in females. Extracellular FXIII molecule has a tetramer structure composing of two catalytic A-subunits and two B-subunits that act as a carrier molecules. Based on genotype there are two types of FXIII deficiency: A-subunit deficiency (XIIIA) when mutations affect F13A gene and much rarely B-subunit deficiency (XIIIB) when mutations affect F13B gene. Both types result in absence of FXIII catalytical activity in plasma. To date, only five families with isolated B-subunit deficiency have been described in the literature. Here we report five novel mutations, affecting F13B gene and resulting in FXIII deficiency. Materials and Methods. FXIII activity was measured by photometric assay according to Fickenscher et al. (1991). Fiveteen exons of F13A gene as well as 12 exons of F13B gene and their flanking regions were amplified by PCR. Sequencing was performed on an automated sequencing system. Results and Discussion. Five german origin patients with mildly reduced FXIII deficiency have been routinely investigated in order to identify genetic defects within F13A and F13B genes. All patients have shown single heterozygous mutations in F13B gene, revealing two missense, one deletion, one insertion, and one splice site mutation. There were no genetic defects in F13A gene. Patient E.M. (female, born 1947, FXIII activity 47–53%, postoperative bleeding) had a missense mutation in exon 2 (c.73 T>C, Cys5Arg) resulting in disruption of disulfide bond between amino acids Cys5 and Cys56. Patient H.D. (male, born 1983, FXIII activity 54%, epistaxis) showed a missense mutation in exon 3 (c.406C>T, Leu116Phe). It may cause instability of disulfide bond between neighbouring Cys117 and Cys71 in the second domain. Patient E.R. (female, born 1989, FXIII activity 53%, increased bleeding during menstruation) was heterozygous for an in-frame deletion (c.471–473delATT) resulting in deletion of Leu138. Patient O.S. (female, born 1983, FXIII activity 46–57%, bleeding after tonsillectomy) had a small insertion (c.1959insT) in the exon 12 changing the distal amino acid (634–641) sequence and predisposing a synthesis of additional 21 new amino acids at the Carboxy-termini of FXIIIB-Subunit due to disruption of Stop codon at position 641. Patient P.G. (male, born 1974, FXIII activity 43%, bleeding after tooth extraction) was heterozygous for a splice site mutation in intron 3 (IVS3-1 G>C). Interestingly this patient turned out also to have a mild von Willebrand Syndrome (FVIII activity 76%, vWF:RiCo 48%, vWF:Ag 56%, vWF-Collagen-Binding activity 0.54, AB0 blood group A). In conclusion, our results suggest higher prevalence of FXIIIB deficiency as it was thought before, Patients having mild (heterozygous) FXIIIB deficiency may bleed upon provocations.

Recent research efforts have provided significant insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). However, the low incidence of the disease hampers the identification of trigger factors initiating an acute TTP episode. In the present work data from 68 TTP patients were analyzed. The patients were asked by a questionnaire to provide data concerning age, gender, medical history, underlying and concomitant diseases and potential triggers. Additional laboratory and clinical data from onset of disease and remission phase were obtained by a second questionnaire completed by the physicians. Our study showed that long term abuse of nicotine is a predisposing factor with an OR of 3.19. The OR for obesity and development of TTP was 2.01. The influence of nicotine abuse has not yet been described whereas the association between obesity and development of TTP is already known. The intake of oral contraceptives (13 of 46 women/28%) of Tamoxifen, of Fluvastatin, of Ticlopidine and of Quinine was identified to be potential triggers. 55% (34 patients) of the patients reported infections and inflammations as triggers (mostly influenza (n=16) and gastroenteritis (n=14)). The relation of female and male patients was 3:1. The mean age, in which the initial TTP episode occurred, was significantly lower in female patients compared with the male patients (33.8 versus 43.8 years). Severe ADAMTS-13 deficiency was found in 91% in all investigated episodes of the patients. Inhibitory activity was associated with severe ADAMTS-13 deficiency in 81% of the cases. The comparison of patients during remission with and without relapses of TTP revealed a significant lower ADAMTS-13 activity in patients with relapsing TTP compared to patients without a relapse of TTP. This result suggests that ADAMTS-13 activity has prognostic value with respect to the occurrence of relapse.

Plasma exchange (PE) is the treatment of choice in thrombotic thrombocytopenic purpura (TTP) patients. The use of PE has decreased the mortality rate of TTP from 90 to 10–30%. However PE is costly, technically demanding and often associated with serious complications such as infection and/or transfusions reactions. Some patients are also unresponsive to PE. In recent studies patients with refractory TTP have achieved sustained remission after Rituximab treatment. Patients with chronic TTP are very often afraid of PE. We cared for 4 anxious patients, who refused PE treatment on the one side and demanded treatment in their early phase of relapse on the other side. Therefore we treated 4 patients with a beginning relapse of TTP with Rituximab (375 mg/m2/week) before onset of symptoms. The 4 patients had already suffered from 10, 3, 4 and 5 relapses. Due to close control of laboratory data in an 8 or 12 weekly interval it was possible to detect deterioration of laboratory data (decrease of platelets <50.000, increase of LDH>500 U/l, decrease of ADAMTS-13 activity to <6.5%, increase of inhibitor to ADAMTS-13 >4BU and detection of schistocytes in the blood smear). Already after the first or second Rituximab dose a significant effect on the laboratory data was seen (increase of platelets >100.000, decrease of LDH to normal values, increase and normalization of ADAMTS-13 activity, decrease of inhibitor titer and disappearance of schistocytes). In the mean observation time (3–12 months) no deterioration of laboratory data and no TTP symptoms were observed. No side effects of Rituximab occurred. Based on our experience we recommend close control of laboratory parameters and an immediate treatment before onset of symptoms in patients with impending relapses. Up to now the reasons for these rapid effects of Rituximab are not fully understood. Other mechanisms of Rituximab treatment in TTP compared to lymphoma therapy might be possible.

Introduction. FXIII deficiency is a rare autosomal recessive disorder affecting approximatly 1 out of 1–3 million inhabitans. The disease is characterized by bleeding, impaired wound repair and spontaneous abortions in females. Extracellular FXIII molecule has a tetramer structure composing of two catalytic A-subunits and two B-subunits that act as a carrier molecules. Based on genotype there are two types of FXIII deficiency: A-subunit deficiency (XIIIA) when mutations affect F13A gene and much rarely B-subunit deficiency (XIIIB) when mutations affect F13B gene. Both types result in absence of FXIII catalytical activity in plasma. To date, only five families with isolated B-subunit deficiency have been described in the literature. Here we report five novel mutations, affecting F13B gene and resulting in FXIII deficiency.

The acquired von Willebrand syndrome (AVWS) is a bleeding disorder, which is amongst others associated with myeloproliferative diseases. Its prevalence varies from 0,04–0,13% in literature. Typical laboratory findings such as a low VWF:RCo/VWF:Ag- Ratio, a prolonged aPTT, PFA and bleeding time, a decrease of VWF:RCo, FactorVIII:, VWF:Ag, typical VWF multimeric structure and the absence of a family history of bleeding are the basis for the diagnosis of AVWS. Bleeding episodes are typical for AVWS and are mostly of the mucocutaneous type (epistaxis, heavy menstrual bleeding, gingival and postoperative bleedings). Myeloproliferative diseases, particularily essential thrombocythemia, are with 15–18% the third most frequent observed comorbidity of patients with AVWS, but the prevalence of AVWS in patients with MPS is -at our knowledge- still unknown. We investigated 54 patients with the diagnosis of myeloproliferative disease (44 ET, 9 PV, 1 IMF), which was established according to the WHO-criteria over a period of 6 years (2000–2006). By analysing the typical laboratory parameters for AVWS, the VWF multimeric structure and the own and family history of bleeding we detected that 67% (36/45) of these patients had an AVWS: 27/44 ET (61%), 8/9 PV (89%), 1/1 IMF. The two most sensitive parameters in this context were VWF:RC/vWF:Ag- Ratio and VWF:RCo. It is well known that AVWS can disappear by treating the MPD. We could also show that treating the MPD with Hydroxycarbamid platelets, VWF multimeric structure, VWF:RCo/VWF:Ag-Ratio and VWF:RCo and the bleeding tendency were normalized. The JAK2-V617F mutation is frequently found in myeloproliferative disorders (MPD): Up to 97% patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) carry this mutation. A further goal in this investigation was to find out a correlation between JAK2-V617F mutation, MPD and AVWS. JAK2-V617F diagnosis was performed in 27 of the 54 patients with MPD. 10 of these 27 patients carried a mutated JAK2-allele. However we observed no significant difference between mutated and unmutated patients in correlation to AVWS. In our investigation the high prevalence of 67% AVWS in patients with MPD confirm the hypothesis that until now AVWS is an underdiagnosed disease. Because of this high prevalence, the possibility of regression by treating MPD and the importance of early diagnosis for prevention of unexpected, sometimes letal bleeding complications, it would be benefical to introduce the AVWS- diagnosis-procedure as a routine step for patients with MPD.

Introduction: Acquired haemophilia (AcH) is associated with the development of polyclonal autoantibodies against FVIII, which affect directly the A2 or C2 domain of the FVIII molecule. Immunomodulatory therapy regimes to normalize FVIII levels and to eliminate the inhibitor are essential options in the treatment of patients (pts) with AcH. The aims of the present study were to investigate the response to Rituximab® and to localize the inhibitor epitopes on the FVIII domains. Patients and Methods: In 5 pts with AcH (2 females,3 males; age: 64–81 yrs) the inhibitor titers ranged from 9 to 156 BU and the FVIII activities from <1 % to 6 %. Rituximab® was administered once weekly for 4 weeks (dosage: 375 mg/m2) in combination with a prednisone therapy (1–1.5 mg/kg). Random peptide phage display libraries were used to identify peptide sequences specific for inhibitors. The short peptide sequences allow the mapping of conformational epitopes on the surface of the FVIII molecule. Results: In all pts (median follow up of 24 months, range: 2–49 months) we documented complete remission (CR). The median time to achieve CR was 13 weeks (range: 2–32 weeks). The time to CR was much longer (23/32 weeks) in two pts, who showed high inhibitor titers (50/188 BU) compared to pts (2/4/5 weeks) with low inhibitor titers (9/22/29 BU). One pt with a high inhibitor titer (50 BU) relapsed 19 months after CR, an other pt died in septic shock 8 weeks after the initiation of therapy with Rituximab®. No other relevant side effects were observed. In the pt with a relapse we identified target epitopes in the A1 and A2 domain of FVIII, and in the other pts (n=3) the peptide sequences mapped to epitopes on the C2 domain of FVIII. Conclusion: The therapy with Rituximab®shows high response rates and seems to be effective in the elimination of high and low inhibitors against FVIII in pts with AcH. Limited by the small number of pts, further studies are needed to confirm whether the different target epitopes found in patients with (A1/A2) or without a relapse (C2) might have a relevance in the specific immunomodulatory therapy.

Hepatitis C virus (HCV) infection is often observed in hemophilic patients who were treated with non-virus inactivated clotting factor concentrates prior to the mid 1980s. HCV infected patients exhibit an increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Hemophilia cohort studies until 2004 suggest a prevalence of HCC development of 0,1–0,2% in HCV infected hemophilic patients. During the last two years we observed a rapid increase in HCC development in our hemophilia treatment center. In the current study we analyzed our data to investigate the impact of chronic HCV infection in hemophilic patients today. We treated 90 hemophiliacs with chronic HCV hepatitis. Seven of these patients (6,3%) developed a HCC between 2002 and 2005, six of them only between 2004 and 2005. Five patients had severe hemophilia A, one mild hemophilia A, one severe hemophilia B, two of them had co-infections (HIV and HIV+HBV). Three had HCV genotype 1b, three 3a and one unknown. Four had been treated with interferon alpha/peginterferon alone or in combination with ribavirin, only one achieved a virologic response. Two patients refused treatment of HCV infection, one patient was not treated due to HIV co-infection. Their age at HCC diagnosis ranged from 40 to 73 years (median 60), time-course between estimated HCV infection and HCC development ranged between 11 and 35 years (median 22). All patients had developed liver cirrhosis: two Child-Pugh A, one Child-Pugh B and four Child-Pugh C. Six patients were asymptomatic; only one patient had jaundice when HCC was detected. All were regularly checked at least once a year with routine screening of AFP level and ultrasound (US) scans. Three patients had normal, four elevated AFP levels (>15ng/ml), three had US scans without pathological finding when HCC was detected. All had consistently or intermittently elevated serum ALT levels during the last five years before diagnosis. HCC was diagnosed either by elevated AFP levels plus US identification (2) or plus MRT (2), by MRT plus check of arterial hypervascularisation (1) plus liver biopsy (1). One patient was only diagnosed after liver transplantation for chronic liver disease. Orthotopic liver transplantation was done in one patient, transcatheter arterial chemoembolization (TACE) in three, two patients have died of liver failure. Our data show that at present HCC is an increasingly frequent cause of mortality in hemophilic patients with chronic HCV infection. The detection of six HCC in only 15 months is striking. Especially nowadays a close follow up on hemophilic patients with chronic HCV infection seems to be mandatory. This should result in earlier detection and consequently more easily treatable tumors and longer survival of our patients.

Von Willebrand’s disease (VWD) is the most common inherited bleeding disorder in humans, exhibiting quantitative and qualitative defects in the von Willebrand factor. To investigate the quality of treatment during the peri- and postoperative course of patients with VWD, we conducted a retrospective data analysis. We analyzed data of 95 patients with VWD who went for 173 surgical interventions in our center between 1981 and 2003. Peri- and postoperative courses with regard of bleeding complications were reviewd. 59 Patients had type1 VWD (two severe, three moderate, 54 mild). 16 patients had type 2 VWD (14 2A, one 2B, one 2N) and 11 patients type 3 VWD. Nine patients with type1 VWD had an additional factor deficiency (five FXII-, two FVII- and two FV-deficiencies). We analyzed the course of 54 orthopedic surgeries, 41 abdominal surgeries, 15 angiographies, 12 endoscopic surgeries, 10 ear-nose-throat interventions, 11 mamma surgeries, 10 neurological surgeries, 8 soft tissue surgeries, 7 thorax surgeries, 3 thyroid gland surgeries and 2 biopsies. Most patients (108/173) were treated with Humate P, 33 patients with DDAVP (33/173) and 9 patients received both products during the pre- peri- and postoperative period. Three patients received other plasmatic FVIII- concentrates, 20 patients had no therapy for their coagulation disorder. 16 patients (9, 2%) had peri- or postoperative bleeding complications. Most of them were seen in patients with VWD type 2B and 3 and in patients who underwent major surgery and who received only DDAVP. Humate P was used for 9 interventions (9/108 = 8, 3%) and DDAVP for four interventions (4/33 = 12, 1%) with bleeding complications, three patients had no therapy. Most of these complications were reported in thoracic surgery (2/7 = 28%), gynecological (3/11 = 27%) and otolaryngological surgery (2/10 = 20%). Retrospectively 15 bleeding complications could be explained by surgical technique, thromboprophylaxis, inefficient substitution therapy or additional thrombocytopenia. In conclusion our findings show a low frequency of bleeding complications in VWS patients with surgical interventions. Our evaluation reveals the necessity of special hemostaseologic care and laboratory controls for these patients. Substitution therapy should depend on type of VWD and type of surgical intervention. Especially during thoracic surgery, gynecological and otolaryngological surgery a careful peri- and postoperative hemostaseologic management as well as careful hemostyptic techniques of the surgeon are required.

Von Willebrand's disease (VWD) is the most common inherited bleeding disorder in humans, exhibiting quantitative and qualitative defects in the von Willebrand factor. To investigate the quality of treatment during the peri- and postoperative course of patients with VWD, we conducted a retrospective data analysis. We analyzed data of 95 patients with VWD who went for 173 surgical interventions in our center between 1981 and 2003. Peri- and postoperative courses with regard of bleeding complications were reviewd. 59 Patients had type1 VWD (two severe, three moderate, 54 mild). 16 patients had type 2 VWD (14 2A, one 2B, one 2N) and 11 patients type 3 VWD. Nine patients with type1 VWD had an additional factor deficiency (five FXII-, two FVII- and two FV-deficiencies). We analyzed the course of 54 orthopedic surgeries, 41 abdominal surgeries, 15 angiographies, 12 endoscopic surgeries, 10 ear-nose-throat interventions, 11 mamma surgeries, 10 neurological surgeries, 8 soft tissue surgeries, 7 thorax surgeries, 3 thyroid gland surgeries and 2 biopsies. Most patients (108/173) were treated with Humate P, 33 patients with DDAVP (33/173) and 9 patients received both products during the pre- peri- and postoperative period. Three patients received other plasmatic FVIII- concentrates, 20 patients had no therapy for their coagulation disorder. 16 patients (9, 2%) had peri- or postoperative bleeding complications. Most of them were seen in patients with VWD type 2B and 3 and in patients who underwent major surgery and who received only DDAVP. Humate P was used for 9 interventions (9/108 = 8, 3%) and DDAVP for four interventions (4/33 = 12, 1%) with bleeding complications, three patients had no therapy. Most of these complications were reported in thoracic surgery (2/7 = 28%), gynecological (3/11 = 27%) and otolaryngological surgery (2/10 = 20%). Retrospectively 15 bleeding complications could be explained by surgical technique, thromboprophylaxis, inefficient substitution therapy or additional thrombocytopenia. In conclusion our findings show a low frequency of bleeding complications in VWS patients with surgical interventions. Our evaluation reveals the necessity of special hemostaseologic care and laboratory controls for these patients. Substitution therapy should depend on type of VWD and type of surgical intervention. Especially during thoracic surgery, gynecological and otolaryngological surgery a careful peri- and postoperative hemostaseologic management as well as careful hemostyptic techniques of the surgeon are required.

Hepatitis C virus (HCV) infection is often observed in hemophilic patients who were treated with non-virus inactivated clotting factor concentrates prior to the mid 1980s. HCV infected patients exhibit an increased risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Hemophilia cohort studies until 2004 suggest a prevalence of HCC development of 0,1–0,2% in HCV infected hemophilic patients. During the last two years we observed a rapid increase in HCC development in our hemophilia treatment center. In the current study we analyzed our data to investigate the impact of chronic HCV infection in hemophilic patients today. We treated 90 hemophiliacs with chronic HCV hepatitis. Seven of these patients (6,3%) developed a HCC between 2002 and 2005, six of them only between 2004 and 2005. Five patients had severe hemophilia A, one mild hemophilia A, one severe hemophilia B, two of them had co-infections (HIV and HIV+HBV). Three had HCV genotype 1b, three 3a and one unknown. Four had been treated with interferon alpha/peginterferon alone or in combination with ribavirin, only one achieved a virologic response. Two patients refused treatment of HCV infection, one patient was not treated due to HIV co-infection. Their age at HCC diagnosis ranged from 40 to 73 years (median 60), time-course between estimated HCV infection and HCC development ranged between 11 and 35 years (median 22). All patients had developed liver cirrhosis: two Child-Pugh A, one Child-Pugh B and four Child-Pugh C. Six patients were asymptomatic; only one patient had jaundice when HCC was detected. All were regularly checked at least once a year with routine screening of AFP level and ultrasound (US) scans. Three patients had normal, four elevated AFP levels (>15ng/ml), three had US scans without pathological finding when HCC was detected. All had consistently or intermittently elevated serum ALT levels during the last five years before diagnosis. HCC was diagnosed either by elevated AFP levels plus US identification (2) or plus MRT (2), by MRT plus check of arterial hypervascularisation (1) plus liver biopsy (1). One patient was only diagnosed after liver transplantation for chronic liver disease. Orthotopic liver transplantation was done in one patient, transcatheter arterial chemoembolization (TACE) in three, two patients have died of liver failure. Our data show that at present HCC is an increasingly frequent cause of mortality in hemophilic patients with chronic HCV infection. The detection of six HCC in only 15 months is striking. Especially nowadays a close follow up on hemophilic patients with chronic HCV infection seems to be mandatory. This should result in earlier detection and consequently more easily treatable tumors and longer survival of our patients.

Introduction: Acquired haemophilia (AcH) is associated with the development of polyclonal autoantibodies against FVIII, which affect directly the A2 or C2 domain of the FVIII molecule. Immunomodulatory therapy regimes to normalize FVIII levels and to eliminate the inhibitor are essential options in the treatment of patients (pts) with AcH. The aims of the present study were to investigate the response to Rituximab® and to localize the inhibitor epitopes on the FVIII domains.

Previously treated patients (PTP), who have not developed an inhibitor (inh.) so far, are considered to be tolerant to factor VIII and at low risk for inh. development. Therefore inh. detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application. To find out about the current situation regarding inh. development in PTP in Germany, we conduct a retrospective study. A questionnaire was sent to 87 haemophilia treating centers, so far 46 of them answered. 28 PTP-inh. developments during the last 5 years were reported. Age of the patient (median 35), severity of haemophilia A (18 severe, 2 moderate, 8 mild), exposuer days (median 30) and gene mutation (intron-22-inversions 31%, large deletions 13%, missense mutations 25%, stop mutation 6%, small deletion 13%) were recorded. 8 different factor VIII concentrates were given during inh. development (5 plasma derived, 3 recombinant). Way of application (16 times bolus infusion, 3 times continuous infusion, 5 times both), former change of product, infused amount until inh. development, inh. characteristic, concomitant diseases and medication were registered. In conclusion most of the patients were over 18 years old (17/28) when the inh. developed. Most of them had severe haemophilia A (18/28) and had at least one change of product before inh. detection. Our data are still too small to draw any conclusion concerning product neo-antigenicity or treatment modalities. Nevertheless it became obvious that inh. development in PTP is still a serious and underestimated problem in haemophilia treatment today. Secondly data showed that there is a variety of PTP definitions in Germany, referring to age of pat, number of exposure days and former change of product. An international definition for PTP would be helpful. A national haemophilia register would make it easier to evaluate data in the future and a prospective, not product related study should be conducted.

We applied peginterferon alfa 2a for off label use therapy in a 44 year old male patient (pat.) with extensive venous malformation (VM) and associated localized intravascular coagulation (LIC). Since birth he had huge VMs in the left leg and left lower abdomen. During surgery and tooth extraction he had severe bleeding complications. In 2003 he reported a sensation of continually growing VMs and requested for therapy. Clinical findings (examination, contrast medium CT) showed no considerable growth of VMs in comparison to former examinations. Laboratory findings were low fibrinogen, low factor VIII activity (FVIII:C) and factor XIII activity (FXIII:C), prolonged aPTT, mild thrombocytopenia and elevated d-dimers. Vascular surgeons disadvised another surgery. LMWH therapy had been carried out before and was ineffective considering coagulopathy and sizereduction of vascular malformations. Considering antiproliferative properties we decided for off label use therapy with peginterferon alfa 2a (Pegasys), 180 μg once a week. Therapy started September 2004 and was applied for 16 weeks. Laboratory tests, clinical examination and photo documentation were done before, during and after therapy. During and after therapy laboratory findings showed normalization of fibrinogen, FVIII:C levels, aPTT and rising FXIII:C levels. We saw no significant change of VMs in clinical examination and photographs. The pat. reported no sense of vascular size reduction but also no sense of growing malformations. Due to interferon side effects such as lowered blood count, therapy was finished after 16 weeks. In conclusion the LIC in the VMs seemed to improve under interferon therapy with normalization of fibrinogen and FVIII:C. Even though there was no significant size reduction of VMs yet, this therapy could be an important treatment option for normalization of coagulation parameters e.g. before surgery. In our patient interferon therapy seemed to be more effective than LMWH therapy. In literature only heparin was reported to be effective in coagulopathies associated with VMs, not interferon. Therefore the positive laboratory findings in our patient are remarkable.

The congenital dysfibrinogenemia is a rare clotting disorder which is based on a structural defect in the fibrinogen molecule. Approximately 350 cases of unrelated families with dysfibrinogenemia have been reported. Of these, the structural defects have been determined in nearly 170 families. Patients and methods: We describe the clinical manifestations in 50 patients from 19 families from Frankfurt. Up to now the underlying structural defect could be found in 43/50 (86 %) of the patients and 14/19 (74 %) of the families (Galanakis, New York, von Depka, Hannover). Results: In 19 families, 50 patients were found with dysfibrinogenemia (52% female, age median: 52 years-old, 9 - 89 years old). The fibrinogen level, according to Clauss decreased in median with 51 mg% (15 – 86 mg/dl, norm: 150 – 450 mg/dl). The determination of the other fibrinogen levels resulted in the normal range. Fibrinogen according to Schulz: 240 mg/dl, immunological fibrinogen: 244 mg/dl. The Reptilase time was prolonged to 55 sec (median, norm: − 20 sec.). 50% of the patients stated a distinct bleeding tendency and mostly a tendency for haematoma (59%) and secondary haemorrhaging (45%). 13 patients had a thrombotic disease, of which 80% were arterially caused. 12 % of the patients stated a tendency for bleeding as well as for thrombosis. 19% of the patients had miscarriages, partly recurrent and 18/50 (36 %) of the patients were asymptomatic. Furthermore, it was observed that the clinical symptoms are often similar in the affected families. By administration of fibrinogen concentrates, the pregnancy in 4 patients who suffered from abortions in the first weeks of pregnancy resulted in healthy children. Fibrinogen was administered from the beginning of the pregnancy till the delivery (aiming fibrinogen levels of Clauss up to 100 mg%). In 11 of the families, an Aalpha 16 Arg-Cys mutation was found. In the other three families there were found gamma mutations, e.g., gamma 356 Ala-Thr, gamma 318–319 del and gamma 318 Ap-Gly. The clinical manifestations in the families with Aalpha 16 Arg-Cys mutation were bleeding (65 %), thrombosis (22 %) and abortions (16 %). 25 % were asymptomatic and 12 % of the patients experienced both, bleeding and thrombosis. Conclusion: The investigations of the Frankfurt patient group with dysfibrinogenemia showed a high rate of clinical manifestations, mostly the tendency for haematoma and secondary haemorrhaging. The tendency for thrombosis is concerned rather with the arterial vessels. The high tendency for miscarriage is striking, which makes the administration of fibrinogen concentrate during pregnancy necessary. The most common found fibrinogen mutation was at the Aaplha chain in position 16. In patients with this mutation mostly both, bleeding and thrombosis occurred. Thus, management of patients with this mutation may be complicated by the variablility of clinical manifestations of congenital dysfibrinogenemia.

Introduction:Major orthopaedic procedures and the presence of thrombophilia are risk factors of thrombotic events. In patients with haemophilia undergoing hip or knee replacement the importance of thrombosis prophylaxis with heparin in the postoperative period is still unknown. The aim of the present study was to evaluate the occurence of thrombotic events in haemophiliacs undergoing major surgery without thrombosis prophylaxis. Patients: A total of 32 pts with haemophilia A (severe:27 pts, moderate:4 pts, mild:1pt; median age:47yrs, range:27–73yrs) undergoing hip (n=9)or knee (n=35) replacement were analysed in our haemophilia treatment center. Pts with inhibitors were excluded. Surgical interventions were performed using recombinant (n=37) or plasma-derived (n=7) FVIII for 12 to 15 postoperative days. The median initial dose of FVIII was 82 IU/Kg−1, followed by median FVIII doses of 54 IU/kg−1over the first four days. All pts received thrombosis prophylaxis with graduated compression stockings only. In addition to factor VIII activity, APC resistance, FV G1691A mutation and the FII G20210A variant (FII) were investigated. Results: No thrombotic events in the postoperative period was dokumented. The median FVIII activity was 153% (range:85–347%), the Body mass index (BMI:kg/m2) was 23.1(range:18.2–30.5). During the first four postoperative days FVIII levels >150% were measured in 24/44 (55%) operative procedures. BMI>25 were shown in 12/32 (38%) pts, and age >40yrs was documented in 23/32 (72%) pts. In 1/30 (3%) pt APC resistance and none of our pts FII were identified. Conclusion: In our study group elevated FVIII levels and additional risk factors (BMI>25, age>40years) seems not to influence relevantly the occurrence of thrombotic events in pts with haemophilia undergoing hip or knee replacement without thrombosis prophylaxis. Further studies are required to confirm whether a thrombosis prophylaxis with heparin is needed in haemophiliacs undergoing high risk surgery.

The plasma metalloprotease ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type I motif - 13) cleaves the Von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1606 and thereby controls the hemostatic activity of the VWF. We tested three commercial VWF concentrates with respect to their ADAMTS-13 activity and antigen contents. ADAMTS-13 activity in the VWF concentrates was tested by measuring the capacity of the concentrates for auto-proteolysis both in the presence and in the absence of neutralising ADAMTS-13 auto-antibodies. To achieve this, the concentrates were reconstituted to a final VWF concentration of 100 I.E. U VWF:Ristocetin Cofactor activity (VWF:RCo). The VWF solutions were diluted with 5 mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples or Imidazole buffer as plasma replacement.The residual VWF:RCo was subsequently measured using the BC von Willebrand Reagent from Dade Behring (Marburg, Germany). The residual VWF:RCo in the presence of plasma from a patient with acquired TTP (ADAMTS-13 activity: <6.25% due to high levels of neutralising ADAMTS-13 auto-antibodies) was then compared to the residual VWF:RCo in the mixtures containing normal plasma, Imidazole buffer or plasma from a patient with congenital TTP (ADAMTS-13 activity: <6.25% due to mutations in the ADAMTS-13 gene). ADAMTS-13 antigen was measured by a commercial ADAMTS-13 ELISA from American Diagnostica (Stamfort, USA). Residual VWF:RCo after proteolysis of VWF concentrates in the presence of Normal plasma, Imidazole buffer and plasma from patients with congenital and acquired TTP, respectively VWF concentrate Normal plasma Imidazole buffer Congenital TTP Acquired TTP A 7% 106% 111% 138% B 10% 11% 16% 104% C 10% 32% 36% 63% ADAMTS-13-Antigen in VWF concentrates given as ng ADAMTS-13 Antigen per I.E. Units VWF:RCo VWF concentrate ADAMTS-13 Antigen [ng/I.E. U VWF:RCo] A not detectable B 6.7 C 0.8 The loss in VWF:RCo in the samples containing either Imidazole buffer or congenital TTP plasma compared to the loss in the samples containing acquired TTP plasma hints at auto-proteolysis due to specific action of ADAMTS-13. All concentrates seem to contain some ADAMTS-13 activity, but ADAMTS-13 activity in the solutions of 100 I.E. U/ml VWF:RCo used here is always lower than the activity in normal plasma (which contain per definition 1U/ml ADAMTS-13 activity). The ratio of ADAMTS-13 activity/VWF:RCo is thus always lower than 1:100 in all the investigated concentrates. However, concentrate B seems to show the highest ADAMTS-13 activity as well as the largest amount of ADAMTS-13 antigen. Concentrate B is followed by concentrate C. Concentrate A only shows traces of ADAMTS-13 activity and non detectable levels of ADAMTS-13 antigen. The results demonstrate that concentrates B and C should not be used as VWF substrates in ADAMTS-13 activity assays. The ADAMTS-13 content in the various VWF concentrates may influence stability, recovery and half-lives of the concentrates.

Continuous infusion (CI) of coagulation factor concentrates has been used in haemophilia treatment since the early 1990s. Recent reports of the occurrence of an inhibitor (inh.) after CI have raised concerns about this method of treatment. We conduct a retrospective study to investigate the development of inh. After CI of factor VIII (FVIII) concentrates in Germany and report on further study results. A questionnaire was sent to 100 haemophilia treating colleagues in Germany. So far 42 colleagues have answered, in 19 haemophilia treatment centers CI was used as treatment modality, 5 of them still use it today. Altogether 200 CI were conducted in 128 patients (pat.). In 14 pat. Inh. Development was detected. 5 of these pat. Were suffering from severe, 1 from moderate and 8 from mild haemophilia, their median age was 27 years. Indications for treatment before inh. Detection were major bleeds and surgical procedures. Plasmaderived (9) and recombinant (4) factor concentrates were given in various infusion sets. Data concerning gene mutation, exposure days, inhibitor characteristic, former change of product, concomitant medication, vaccination and blood transfusion were collected. Regarding the genotype, we found 4 missense mutations, 2 intron-22-inversions, 1 small deletion, 1 splice site mutation, 6 were unknown. In our study strikingly the inh. Developed very often in pat. With mild or moderate haemophilia and genotypes that exhibit an inh. Prevalence of < 10 %. Our further data confirm the assumption that pat. With mild haemophilia might exhibit a much higher prevalence of inh. Development when treated with an “intensive FVIII - treatment” such as CI. On the other hand there might be an uncommon inh. pathomechanism in connection with CI, resulting in a peculiar group of pat. developing the inh. after this way of application. A prospective multicenter study to investigate the inhibitor development after CI should be conducted.

We applied peginterferon alfa 2a for off label use therapy in a 44 year old male patient (pat.) with extensive venous malformation (VM) and associated localized intravascular coagulation (LIC). Since birth he had huge VMs in the left leg and left lower abdomen. During surgery and tooth extraction he had severe bleeding complications. In 2003 he reported a sensation of continually growing VMs and requested for therapy.

Introduction:Major orthopaedic procedures and the presence of thrombophilia are risk factors of thrombotic events. In patients with haemophilia undergoing hip or knee replacement the importance of thrombosis prophylaxis with heparin in the postoperative period is still unknown. The aim of the present study was to evaluate the occurence of thrombotic events in haemophiliacs undergoing major surgery without thrombosis prophylaxis.

The plasma metalloprotease ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type I motif - 13) cleaves the Von Willebrand factor (VWF) at the peptide bond Tyr1605-Met1606 and thereby controls the hemostatic activity of the VWF. We tested three commercial VWF concentrates with respect to their ADAMTS-13 activity and antigen contents. ADAMTS-13 activity in the VWF concentrates was tested by measuring the capacity of the concentrates for auto-proteolysis both in the presence and in the absence of neutralising ADAMTS-13 auto-antibodies. To achieve this, the concentrates were reconstituted to a final VWF concentration of 100 I.E. U VWF:Ristocetin Cofactor activity (VWF:RCo). The VWF solutions were diluted with 5 mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2and different plasma samples or Imidazole buffer as plasma replacement.The residual VWF:RCo was subsequently measured using the BC von Willebrand Reagent from Dade Behring (Marburg, Germany). The residual VWF:RCo in the presence of plasma from a patient with acquired TTP (ADAMTS-13 activity: <6.25% due to high levels of neutralising ADAMTS-13 auto-antibodies) was then compared to the residual VWF:RCo in the mixtures containing normal plasma, Imidazole buffer or plasma from a patient with congenital TTP (ADAMTS-13 activity: <6.25% due to mutations in the ADAMTS-13 gene). ADAMTS-13 antigen was measured by a commercial ADAMTS-13 ELISA from American Diagnostica (Stamfort, USA).

Previously treated patients (PTP), who have not developed an inhibitor (inh.) so far, are considered to be tolerant to factor VIII and at low risk for inh. development. Therefore inh. detection in a PTP should raise concerns about the concomitant variables such as product neo-antigenicity or way of application.