bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of rVWF with Polysialic Acid: Biochemical and Functional Characterization in Mice with VWD.
Authors: Turecek Peter L. , Siekmann Juergen , Weber Alfred , Gritsch Herbert , Váradi Katalin , Matthiessen H. Peter , Muchitsch Eva-Maria , Baumgartner Bernhard , Schwarz Hans Peter 
Published: April 2020

Abstract
Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we tried to introduce additional sialic acids on rVWF to obtain the opposite: prolonged survival of rVWF in the circulation. Additional sialic acid residues were introduced to rVWF by chemically attaching polysialic acid (PSA, colominic acid) to lysines on recombinant VWF. rVWF was purified from the cell culture supernatant of CHO cells co-expressing rFVIII and rVWF. Coupling of PSA was performed by terminal oxidation of the vicinal diols in PSA to aldehydes and binding to the primary amino groups in the VWF molecule by Schiff-base formation. Schiff-bases were stabilized by reduction with NaCNBH3 under formation of a secondary amine.
PSA-rVWF conjugates retained their multimeric structure but had slightly increased molecular sizes of the VWF sub-units due to bound PSA. The attachment of PSA to rVWF resulted in a decrease of RCo activity and a decrease in FVIII binding capacity to approximately 60%.
Administration of PSA-rVWF at a dose of 100 units rVWF antigen per kg to VWF-deficient mice resulted in a 2-fold increase in the survival of rVWF as compared to non-modified rVWF. A secondary rise in endogenous murine FVIII after substitution of VWD mice with human VWF occurred elevating FVIII levels from 0.1 (baseline) to 0.4 IU FVIII/mL at 3 hours after application of both non-modified and polysialylated rVWF. Maximum levels of ~ 0.4 IU FVIII/mL were maintained for more than 20 hours in the group treated with PSA-rVWF despite the reduced FVIII binding capacity observed in vitro, while FVIII levels in mice treated with non-modified rVWF steadily declined and returned to their baseline level after 21 hours. The FVIII AUC was 5.3 and 3.3 IU/mL/hr for mice treated with PSA-rVWF and non-modified rVWF, respectively.
We conclude that polysialylation of rVWF represents a method to prolong the survival of rVWF in the circulation.
A Limited Number of Anti-Idiotypic Antibodies Is Required To Maintain FVIII Function in Presence of Polyclonal Anti-FVIII Antibodies of Haemophilia A Patients with Inhibitors.
Authors: Gilles Jean Guy G. , Grailly Sabrina E. , Jacquemin Marc G. , Saint-Remy Jean Marie M. 
Published: April 2020

Abstract
Replacement therapy using either plasma-derived or recombinant FVIII elicits FVIII-specific Abs in about 25% of the patients. The majority of such antibodies are directed towards specific FVIII regions on which major epitopes have been identified. Most of these epitopes are located in the carboxy-terminal end of the C2 domain of the FVIII light chain and in the amino-terminal end of the A2 domain of the heavy chain, whereas, antibodies directed towards the C1 domain have been described in cases of mild/moderate haemophilia A. From a functional point of view, epitopes have been mapped to the phospholipid and von Willebrand binding sites in the C1 and C2 domains and to the FIX binding site in A2.
We have derived human mabs (mabRhd5, mabBo2C11 and mabBoIIB2) that react with high affinity to the FVIII C1, C2 and A2 domains, respectively, and are representative of most of the specific inhibitors observed in haemophilia A patients.
Recently, we have generated mouse anti-Id mab (mab14C12) against mabBo2C11 and provided the first demonstration that an anti-Id mab directed towards a C2-specific inhibitor restored FVIII activity both in vitro and in vivo in the presence of the inhibitor. Futher more, we demonstrated that FVIII inhibition obtained by a mixture of two anti-FVIII mabs (mabBo2C11 (anti-C2) and mabBoIIB2 (anti-A2)) was neutralized up to 100% when a mixture of the corresponding anti-Ids (mab14C12 and mab30D1) was added to the assay establishing the proof of concept that combination of anti-Id mabs will be able to neutralize the anti-FVIII immune response of haemophilia A patients. Based on this observation, We have also generated an anti-idiotypic antibody (mab18B6) towards an anti-C1 domain inhibitor (mabRHD5). In ELISA, mab18B6 specifically prevents, in a dose-dependent manner, mabRHD5 binding to C1 and fully neutralized mabRHD5 inhibitory properties in a coagulation assay. With a mixture of anti-Ids (mab14C12, mab30D1 and mab18B6) we were able to completely neutralized the cumulative anti-FVIII activity obtained in functional assay by a mixture of the corresponding monoclonal anti-FVIII antibodies. We next evaluated whether a combination of anti-Ids (anti-anti-A2,-C1,-C2) had the ability to neutralize the inhibitory properties of polyclonal antibodies obtained from haemophilia A patients. To this end, eight patients with severe haemophilia A and inhibitor were tested. An average of 70% neutralization of the inhibition was obtained in 6 out of 8 cases. These data allow to conclude that potent polyclonal high-affinity FVIII inhibitors can be neutralized with corresponding anti-idiotypic antibodies and that only a limited number of anti-idiotypic antibodies is required for inhibitor neutralization in a majority of patients.
Factor XI Deficiency Confers a Survival Advantage in a Murine Sepsis Model.
Authors: Gailani David , Tucker Erik I. , Cheng Qiufang , Hanson Stephen R. , Gruber Andras 
Published: April 2020

Abstract
Bacterial sepsis is frequently accompanied by intravascular activation of coagulation and fibrinolysis, resulting in a coagulopathy with thrombotic, hemorrhagic, and inflammatory components (disseminated intravascular coagulation - DIC) that contributes significantly to morbidity and mortality. Recently, using vascular occlusion models, the plasma coagulation protease factor XI (FXI) has been shown to contribute to pathologic thrombus formation in primates, mice, and rabbits. These results are supported by epidemiologic studies in human populations that show an association between elevated plasma FXI levels and an increased risk for venous and arterial thrombosis. FXI has also been shown to contribute to the lethal consumptive coagulopathy in protein C deficient mice. To investigate the contribution of FXI to consumptive coagulopathy and mortality in sepsis, we compared wild type (WT) and FXI deficient (FXI−/−) mice in models of polymicrobial sepsis and endotoxemia. Polymicrobial sepsis was induced in WT and FXI−/− C57Bl/6 mice (n = 24 for each genotype) using an established model of cecal ligation and puncture (CLP). Overall survival was 21% for WT and 42% for FXI−/− mice (p &lt; 0.01), with WT deaths occurring between days 2 and 4, and FXI−/− deaths between days 3 and 6. Average survival for mice succumbing to sepsis was 55 ± 10 hr for WT, and 88 ± 22 hr in FXI−/− mice (p &lt; 0.01). 24 hrs after CLP, platelet count in WT and FXI−/− animals decreased 27 ± 13% and 13 ± 12% (p = 0.05), and leukocyte counts decreased by 64 ± 13% and 40 ± 23% (p &lt; 0.01), respectively. Fibrinogen levels 24 hours post-CLP decreased slightly in WT mice (37 ± 34% from baseline), while increasing 212 % ± 74% in FXI−/− mice (p &lt; 0.01). Mice were also challenged with intraperitoneal Escherichia coli endotoxin. Mortality 24 hrs after endotoxin administration was identical for WT and FXI−/− mice (n =15 for each dose) at doses of 50 mg/kg (mortality 80%) and 25 mg/kg (mortality 70%). Twelve hours after administering 25 mg/kg endotoxin, the mean platelet counts in WT animals decreased by an average of 60% (from 440 ± 159/mm3 at baseline to 175 ± 77/mm3, n = 10), and by 36% in FXI−/− animals (from 474 ± 218/mm3 to 303 ± 100/mm3). In summary, prognosis was better for FXI−/− mice in CLP-induced sepsis compared to WT mice, and the CLP and endotoxin data indicate that consumptive coagulopathy is less severe in the absence of FXI. The results suggest that therapeutic inhibition of FXIa activity could be beneficial in treating sepsis-related DIC. Humans with severe congenital FXI deficiency typically have, at most, a mild trauma-induced bleeding disorder. It is possible, therefore, that inhibition of FXIa in DIC may be associated with a lower risk of exacerbating bleeding when compared to anticoagulant therapies such as heparin or activated protein C.
Establishment of ES Cells Secreting Human Factor VIII for Hemophilia A-Targeted Cell Therapy.
Authors: Kasuda Shogo , Sakurai Yoshihiko , Kubo Atsushi , Shima Midori , Keller Gordon , Yoshioka Akira 
Published: April 2020

Abstract
Hemophilia A is an X-linked bleeding disorder resulting from abnormality in the coagulation factor VIII (FVIII) gene. Although bleeding episodes can be prevented or treated by intravenous injection of FVIII concentrates, there are some drawbacks such as necessity of frequent intravenous transfusion and high cost of FVIII concentrates. Lots of basic studies for gene therapy have been reported but clinical trials for hemophilia have been interrupted by significant safety concerns. Cell-based therapy may overcome the disadvantages, without impairing the advantages of gene therapy. Therefore, we have tried to use embryonic stem (ES) cells for production of FVIII protein. For the stable expression of FVIII, we established tet-hFVIII ES cells, which induce human (h) FVIII mRNA (F8) by doxycycline (Dox) exposure. Tet-hFVIII ES cells were cultured in the serum containing and/or serum free media and then FVIII activity (FVIII:C) and FVIII antigen (FVIII:Ag) were measured in the supernatant by APTT-based FVIII:C assay and ELISA, respectively. In the previous study, we demonstrated that ES cells could be differentiated with serum into cells with mesoderm potentials such as hematopoietic and vascular endothelial cells. Although mRNA of hFVIII was detected by Dox induction, neither FVIII:C nor FVIII:Ag was detected in the conditioned media in undifferentiated ES cells and differentiated cells with mesoderm potentials. Next we cultured tet-hFVIII ES cells using endoderm and hepatocyte differentiation condition in which differentiated ES cells express Foxa2 and albumin genes (Kubo et al. Development 2003), since FVIII is known to be produced in the liver. Both FVIII:C (3.7 mIU/ml/mg protein) and FVIII:Ag (0.3 ng/ml/mg protein) were detected in the conditioned media for hepatocyte differentiation. These data demonstrated that ES cells could produce and secrete the functional hFVIII protein in our system, when ES cells cultured in the hepatocyte condition. The level of FVIII:C and FVIII:Ag were increased twice by addition of von Willebrand factor (2.5 μg/ml) into the culture media. Furthermore, Dox enhanced the level of FVIII:C and FVIII:Ag dose-dependently. In conclusion, we confirmed secretion of hFVIII in endodermal differentiation culture system. This system may be applicable for cell-based therapy targeting to hemophilia A.
Novel Large Deletion Is the Most Frequent Cause of Type 3 von Willebrand Disease in Hungary.
Authors: Mohl Adrien , Masszi Tamás , Nagy Eszter , Obser Tobias , Oyen Florian , Sallai Krisztina , Bodo Imre , Schneppenheim Reinhard 
Published: April 2020

Abstract
Background: Type 3 is the most severe form of von Willebrand disease (VWD) caused by the virtual absence of von Willebrand factor (VWF) in affected patients. The prevalence of type 3 VWD in Hungary is 2.6 per million. Capitalizing on a nationwide National Bleeding Disorder Registry, we designed a study to characterize the genetic background of the entire Hungarian type 3 VWD population. The current report focuses on the molecular characterization of a novel large deletion.
Methods: 24 patients from 23 unrelated families were studied by direct sequencing of the 52 exons of the VWF gene. The breakpoints of a large deletion were characterized by standard gene mapping. Breakpoint-specific PCR was used to confirm the presence of the deletion, and to screen for identical deletions in other populations from Germany, Russia, and Poland.
Results: A large partial deletion (delExon1-3) of the 5′-region of the VWF gene was detected in 10 alleles (19 percent of all type 3 mutations). Five patients from 4 unrelated families were homozygous, and 2 patients were heterozygous for the deletion. Consanguinity was known in one of the families. In comparison, 2435 delC in exon 18, a common cause of type 3 VWD in some European populations, was found on 6 alleles (12 percent; one patient homozygous). The large deletion resulted in the loss of a 35 kb fragment, incorporating exons 1, 2 and 3. The 5′ breakpoint is located in the 5′ untranslated region, while the 3′ breakpoint is in intron 3 of VWF. No other known gene is lost with the deletion. Clinically, all homozygous patients had serious bleeding episodes from infancy requiring frequent VWF substitutions. However, bleeding became much milder in all patients with no significant spontaneous bleeding after the age of 3-5 years. No inhibitor to VWF was detected. delExon1-3 was not detected in any of the other screened populations.
Conclusion: We report a large novel deletion including exons 1, 2 and 3 of VWF commonly causing type 3 VWD in the Hungarian population. This mutation, which is most probably due to a founder effect, seems to be unique to Hungarian patients with a high allele frequency. Together, delExon1-3 and 2435delC make up 31 % of genetic defects in Hungarian patients with VWD type 3. This offers a rational approach to molecular testing of respective families in Hungary.
Galectin-3 Binding Protein Contaminates the Purification of Recombinant Factor IX.
Authors: Blostein Mark D. , Cuerquis Jessica , Galipeau Jacques 
Published: April 2020

Abstract
The hemophilas are the most common severe hereditary bleeding disorders. The standard treatment of these disorders is to infuse plasma concentrates of the missing coagulation factor. However, the high cost of replacement therapy and the serious morbidity from the transmission of infectious agents have mandated a need for alternative therapeutic strategies. One of these strategies is gene therapy and we embarked on a novel gene therapeutic strategy involving the use of factor IX-transduced bone marrow stroma to treat hemopihlia B, a deficiency of the gamma-carboxylated protein, factor IX. The rate limiting step for the production of factor IX is gamma-carboxylation, a post translational modification carried out only in mammalian cells. In order to test the carboxylation efficiency of our gene therapeutic platform, we first transfected Human Embryonic Kidney (HEK) 293 cells with a retroviral construct containing factor IX. Factor IX was purified by immunoaffinity chromatography and then subject to hydroxyapatite chromatography to isolate biologically active properly carboxylated factor IX from inactive uncarboxylated factor IX. Unexpectedly, during hydroxyapatite chromatography, we discovered that purified factor IX was contaminated by a heretofore unknown protein. Further analysis by mass spectrometry sequencing revealed this protein to be galectin-3 binding protein (G3BP).G3BP contaminates media of HEK293 cells regardless of whether factor IX is transfected into these cells. Furthermore, the contamination of purified media is not specific for factor IX as G3BP is isolated regardless of the antibody used for purification. In summary, the above results provide important information regarding the production of recombinant factor IX and uncover a potentially new contaminant in recombinant products produced in mammalian cells.
The Genetics of Hemophilia: Analysis of Patients at the Hospital for Sick Children, Toronto, Canada.
Authors: Knox David , Samuel Christopher , Pazmino-Canizares Janneth , Barnes Chris , Floros Georgina , Stain Ann Marie , Carcao Manuel 
Published: April 2020

Abstract
There is great interest in identifying genetic mutations responsible for hemophilia and in determining if/how mutations correlate with disease phenotype. A hemophilia genetic database was created at SickKids in 2004. At the time &lt;40% of hemophiliacs followed by the clinic had been genotyped. Currently mutations have been identified on 194/236 patients (82%) followed in the clinic. From this we are performing genotype/phenotype correlations. Preliminary analysis has revealed the following novel findings:
Most mothers of hemophiliacs are carriers; even when there is a no family history of hemophilia. Of the 199 mothers of the 236 children only 6 have been shown not to be carriers; 205 have been shown to be carriers; 15 have not been tested and 10 are unavailable for testing. We believe that most de novo FIX and FVIII mutations occur in females. Mutations responsible for hemophilia B show poor concordance with disease severity i.e. for any mutation the disease severity is not always the same. One example found in 10 hemophiliacs (5 families) who despite having the same missense mutation in exon H have shown FIX levels anywhere between 0 and 11%. Given that disease severity is assigned according to factor levels these patients have been labeled as either severe (n = 3), moderate (n = 5) or mild (n = 2). Clinically these patients all appear to behave as moderate. This points to the fallibility of using FIX levels (which varies according to patient age and health state) in labeling patients. Other aspects of the hemophilia genetic data base are being analyzed. We believe that a detailed study of the genetics of hemophilia will point to novel findings that will eventually translate into patient care.
Defibrotide Does Not Cross-React with HIT Antibodies. Implications in the Management of HIT.
Authors: Fareed Jawed , Hoppensteadt Debra A. , Iacobelli Massimo , Walenga Jeanine M. 
Published: April 2020

Abstract
Defibrotide is a mammalian DNA derived antithrombotic and anti-ischemic agent that does not produce systemic anticoagulation. Heparin-induced thrombocytopenia (HIT) is an immune disease related to heparin exposure, in which patients are at risk of developing life- and limb-threatening thrombosis. Studies were performed to determine whether defibrotide cross-reacts with HIT antibodies. Sera from 141 clinically confirmed HIT patients were tested for platelet activation in the presence of defibrotide (1 – 100 μg/ml) and unfractionated heparin (1 – 100 μg/ml). 103 sera (73%) produced platelet aggregation and serotonin release activities with heparin. Only 4 samples (2%) showed a weak reactivity with defibrotide which was eliminated by heparinase treatment indicating heparin contamination in the patient sample. Further studies revealed that defibrotide does not complex with platelet factor 4 (PF4), and that prolonged incubation of defibrotide with platelet rich plasma does not mobilize PF4. Studies of patients treated with extended dosing of intravenous or oral defibrotide (n=270) demonstrated that HIT antibodies are not generated with defibrotide treatment. Studies carried out on the effect of purified IgG from HIT patients revealed that defibrotide blunts platelet activation and microparticle formation as measured by flow cytometry. These studies suggest that defibrotide may be a useful antithrombotic agent for the management of patients with HIT. Moreover, unlike heparin, defibrotide does not promote platelet activation, rather it is capable of suppressing the hypercoagulable state associated with HIT. Defibrotide is orally bioavailable and can be used for extended anticoagulant management of HIT patients.
Pancreatitis Preceding Acute Episodes of Thrombotic Thrombocytopenic Purpura: Report of Five Patients with a Systematic Review of Published Reports.
Authors: Swisher Karen K. , Doan John T. , Vesely Sara K. , Kwaan Hau C. , Kim Benjamin , Lammle Bernhard , Hovinga Johanna Kremer , George James N. 
Published: April 2020

Abstract
Thrombotic thrombocytopenic purpura (TTP) is a syndrome with multiple recognized etiologies, associated conditions, and risk factors but the events that may precipitate acute episodes are unclear. Patients may have undetectable ADAMTS13 activity without evidence for TTP until a precipitating condition, such as pregnancy, infection, or surgery, occurs. These clinical observations are similar to experimental observations on transgenic mice with absent ADAMTS13 in which a TTP-like condition only occurred when an additional stimulus, Shiga toxin or collagen/thrombin, was administered. Also patients may have acute episodes of typical TTP without apparent ADAMTS13 deficiency. We report 5 patients (Table) who had the onset of an acute episode of TTP 1–13 days following the onset of pancreatitis. Three patients are from The Oklahoma TTP-HUS Registry, a series of 360 consecutive patients with TTP, 1/1/1989 – 6/30/2006. Two patients are from Northwestern University. In each patient there was an apparent etiology for the pancreatitis: bile duct obstruction in patients 1, 4 and 5 and alcoholism in patients 2 and 3. There was no evidence for TTP when the pancreatitis was diagnosed. In patients 1 and 2, the pancreatitis was clearly resolving when the TTP occurred. ADAMTS13 was measured in patients 2–5 and was severely deficient with a high-titer inhibitor in patients 3 and 5. Patient 5 had a recurrent episode of pancreatitis that was also followed by an acute episode of TTP.
No. Laboratory data on days of diagnosis of pancreatitis and of TTP Hgb (g/dL) Plt (103/μL) Cr (mg/dL) LDH (U/L) Amylase (U/L) Days1 (Pan-TTP) (Pan-TTP) (Pan-TTP) (Pan-TTP) (Pan-TTP) 1 Days between onset of acute pancreatitis and diagnosis of TTP 1 6 12.8–8.0 346–40 0.9–2.1 272–1526 1077–154 2 5 14.3–9.0 291–24 0.7–7.7 345–1668 1704–140 3 3 15.4–10.1 301–53 1.2–2.0 --1093 140— 4 1 12.5–9.6 359–74 1.5–4.1 --1195 --- 5a 13 10.3–8.0 588–34 0.8–2.1 --997 627–433 5b 7 13.8–10.0 340–45 1.3–1.1 --541 1584–40
A systematic review of published case reports identified 16 additional patients who had TTP following pancreatitis. Seven patients were alcoholic; 5 patients had gallbladder disease. Coagulation tests were reported in 12 patients and all were normal. Recurrent TTP following a recurrence of pancreatitis was reported in 3 of the 16 patients. Our 5 patients and the 16 previously reported patients all recovered from TTP.
CONCLUSION: Pancreatitis, a disorder that results in an intense systemic inflammatory response, may provoke acute episodes of TTP. Inflammatory cytokines may precipitate acute episodes of TTP by stimulating endothelial cell release of ultralarge von Willebrand factor multimers and inhibiting the cleavage of these multimers by ADAMTS13.