ALBERT

Beschreibung: The authors provide evidence of a novel association between transcription factor IKZF5, one of the IKAROS family proteins, and thrombocytopenia with decreased alpha granules, thus significantly extending our understanding of the gene defects leading to inherited thrombocytopenia.

Beschreibung: Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs 〉0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.

Beschreibung: Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Beschreibung: Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Beschreibung: Key Points Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders. The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.

Beschreibung: Key Points A gain-of-function variant in DIAPH1 causes macrothrombocytopenia and hearing loss and extends the spectrum of DIAPH1-related disease. Our findings of altered megakaryopoiesis and platelet cytoskeletal regulation highlight a critical role for DIAPH1 in platelet formation.

Beschreibung: Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

Beschreibung: Background: Splanchnic vein thrombosis (SVT) includes extrahepatic portal vein obstruction (EHPVO), Budd-Chiari syndrome (BCS) and mesenteric vein thrombosis. Myeloproliferative neoplasms (MPN) account for the majority of non-cirrhotic SVT and are diagnosed in 30% of patients with EHPVO and 40% with BCS. Recurrent thrombosis is a common and significant complication in patients with PVT and MPN. Causes of thrombosis in this patient group are unknown; it is likely multifactorial, with proposed risk factors including increased platelet mass and platelet hyper-reactivity. There is evidence that in general, patients with MPN have increased platelet reactivity when compared to healthy controls, but, there is no data looking specifically at patients with SVT. However patients with SVT clearly demonstrate a significantly more aggressive phenotype in terms of systemic thrombosis, with risk of recurrent thrombotic episodes. MASCOT is a multicenter observational study assessing morbidity and portal circulation in patients with SVT, in a subset of whom we have assessed platelet activity. Methods: Whole blood flow cytometry was used to assess platelet activity by surface expression of P-selectin (CD62P) an established marker of platelet activity. We measured CD62P at baseline in unstimulated platelets and following stimulation with increasing concentrations of thrombin receptor activator protein (TRAP), range 10-50μmol. The guidelines from the European consensus on platelet flow cytometry were used for this assay (Schmitz G et al 1998). Demographic and clinical data were collected. Results were analysed in GraphPad Prism using t-tests; all values displayed as mean (95% confidence interval). Results: We assessed platelet activity in 14 patients with SVT using healthy controls (n=6) for comparison. In our patient cohort 8/14 (57%) were male and 6/14 (42%) female. The average age of our patients was 49 years. 12/14 (85%) were positive for the JAK2 mutation and 2/14 (15%) had a positive calreticulin mutation (CALR+). 2/14 (14%) patients had myelofibrosis (MF), 6/14 (42%) polycythaemia vera (PV), 4/14 (28%) essential thrombocytosis (ET) and 2/14 (14%) were positive for the JAK2 mutation but did not show evidence of MPN in their bone marrow and had normal blood counts. 10/14 (71%) were on warfarin alone, 2/14 (14%) on warfarin and aspirin, 1/14 (7%) on rivaroxaban and 1/14 (7%) on aspirin alone. Platelet activity as measured by CD62P expression was increased in patients with SVT at baseline compared with controls 13.1% (5.5-20.7) vs 0.5% (0.2-0.7) (p=0.003). Platelet activation and reactivity was significantly greater in the patient group compared with controls at all concentrations of TRAP (figure 1). Control platelets were relatively unreactive to trap stimulation and a significant increase from baseline in CD62P at the highest concentration of TRAP used (50 μmol) 0.5% (0.2-0.7) vs 7.31% (1.17-13.4) (p=0.03), however this is a modest increase, the biological significance of which is uncertain. In the patient population, the platelets were hyper-reactive to TRAP induced up-regulation of CD62P. This was significant when comparing unstimulated platelets at the 25 and 50 μmol concentrations 13.1% (5.5-20.7) vs 22.6% (13.8-39.4) (p=0.0008) and 13.1% (5.5-20.7) vs 34.9% (22.6-47.1) (p

Beschreibung: This paper reports on the use of a high-throughput diagnostic genetic screening for coagulation, platelet, or thrombotic disorders in a series of more than 2000 patients.

Beschreibung: Abstract 3160 Poster Board III-97 The major challenges in acquired bleeding disorders are accurate assessment of bleeding risk and subsequent effective correction of the haemostatic imbalance without increasing the risk of thrombotic complications. The response to Fresh Frozen Plasma (FFP) is extremely variable when used in acquired bleeding disorders. The administration of higher doses of FFP is more effective (Chowdhury et al., Br J Haematology. 2004 Apr; 125(1):69-73), but is limited by the potential risk of fluid overload. In addition, as with the use of all blood products, the risk of transmitting infection and concerns regarding allergic reactions, anaphylaxis and transfusion-related lung injury remain. With the increasing availability of prothrombin complex concentrates (PCCs), it has become possible to give a defined dose of factor II, VII, IX, X and proteins C & S in a small volume. Its efficacy has been well described in patients requiring warfarin reversal, but the indication for its use in other acquired bleeding disorders is less clear and data published on the subject is very limited. Concerns remain regarding the thrombotic risk associated with its use and, as a plasma-derived product, there is also the potential of transmitting blood borne infections. Here we present our two-year experience of the use of PCCs in a wide range of acquired coagulopathies. We collected retrospective data on a total of 200 (n=200) administration events, which we subdivided as follows: (1) Warfarin reversal (n=54), (2) Gastrointestinal bleeding on the background of coagulopathy secondary to chronic liver disease (n=62), (3) Liver transplantation complicated by massive blood loss or renal impairment requiring volume restriction (n=31), (4) Massive haemorrhage (n=10), (5) Coagulopathy due to underlying malignancy (n=10), (6) Correction of coagulopathy prior to procedures (n=20), (7) Miscellaneous (n=13). Dosing of PCCs was decided based on weight of patient, severity of acquired coagulopathy, rate of blood loss and underlying pathology. If required the PCCs were administered in conjunction with fibrinogen concentrate, blood products (packed red cells, platelets, FFP, cryoprecipitate) and antifibrinolytic agents such as aprotinin or tranexamic acid. Our PCC dosing regime of 20 -30 units/kg usually led to an improvement in prothrombin time (PT) and international normalised ratio (INR) in all the above listed clinical settings. No excessive blood loss was noted during the procedures for which PCCs were given. In addition, we did not see a significant increase in thrombotic complications in our cohort of patients, but these findings have to be confirmed in larger studies. At our institution, patients requiring warfarin reversal with an INR 〉4.0 receive a universal dose of 30u/kg of PCCs based on a previous study (Gatt et al., J. Thrombosis and Haemostasis. 2009 Jul; 7(7); 1123-7), which demonstrated that sufficient thrombin generation is achieved with this dose and the retrospective analysis of our clinical data supports this. In patients with acute and chronic liver failure, the capacity to generate thrombin is probably not significantly impaired in steady state despite a prolonged PT and INR (Gatt et al., Blood, vol.112, No. 11, abstract 1826). However, these patients are more vulnerable to disturbances caused by major blood loss requiring large volume transfusion. We found that administration of PCC in this setting led to improvement of prothrombin time/INR and clinical outcome. We were able to correlate these findings to improvement in the thromboelastogram curves in patients undergoing liver transplant. We conclude that use of PCCs and their dosing must be tailored to the clinical setting. Administration of PCCs can be considered when rapid correction of clotting factors to haemostatic levels is required and/or if there is a significant restriction to volume that can be safely transfused. A comprehensive approach to the management of acquired coagulopathies should also include the addition of FFP, cryoprecipitate, fibrinogen concentrate, antifibrinolytics and recombinant factor VIIa. The correlation between bleeding tendency and standard coagulation tests remains unclear and has to be further investigated to help with future changes of clinical practice. Disclosures No relevant conflicts of interest to declare.