bluuebunny/crossref_metadata_2025 huggingface dataset:
High Risk of Pregnancy Related Venous Thromboembolism in Women with a Hereditary Deficiency of Antithrombin, Protein C or Protein S in Concomitance of Other Thrombophilic Defects: A Retrospective Cohort Study.
Authors: Folkeringa Nienke , Brouwer Jan-Leendert P. , Korteweg Fleurisca J. , Veeger Nic J.G.M , Erwich Jan Jaap H.M. , van der Meer Jan
Published: October 2019

Venous thromboembolism (VTE)during pregnancy is still an important cause of maternal morbidity and mortality in the Western world. The risk VTE in pregnant women is five times higher than in non-pregnant women of the same age. This risk is increased by thrombophilic defects. In a family cohort study we assessed the risk of VTE during pregnancy/puerperium and the contribution of concomitant thrombophilic defects in women with hereditary antithrombin, protein C or protein S deficiencies.

Women were recruited from a previous large family cohort study, which was designed to estimate the risk of VTE in first degree relatives from probands with documented VTE and a deficiency of antithrombin, protein C or protein S. Probands were excluded from analysis. Blood samples of female relatives were tested on the index deficiencies and prothrombin G20210A, factor V Leiden, hyperhomocysteinemia and increased levels of VIII:C, IX:C and XI:C. Observation time was defined as the fertile period (from age 15–45 years), or until the first VTE, or until end of study period.

The cohort contained 222 female relatives, of whom 101 women were deficient and 121 non-deficient. The absolute risk of venous thromboembolism in deficient women was 1.76 per 100 person-years versus 0.19 per 100 person-years in non-deficient women (Relative Risk (RR) 9.2; 95% CI 3.8–26.7). Concomitance of none, one and more than one thrombophilic defect increased the risk in deficient (1.55, 2.14 and 2.92 per 100 person-years, respectively) and non-deficient women (0.16, 0.09 and 0.54 per 100 person -years, respectively). Annual incidences (AI) were lower in ever pregnant than never pregnant deficient women, 1.37 per 100 person-years versus 2.96 per 100 person-years (RR 0.5; 95% CI 0.2–0.99)(Table). These were 0.09 per 100 person-years versus 0.70 per 100 person-years in non-deficient women (RR 0.1; 95% CI 0.02–0.9). In ever pregnant deficient women 71% of VTE episodes were pregnancy related. A majority (75%) of never pregnant deficient women revealed VTE associated with oral contraceptives.

Women with hereditary antithrombin, protein C or protein S deficiencies are at high risk of VTE. Concomitance of other thrombophilic defects increased this risk. In fertile women most episodes of VTE are associated with either pregnancy or oral contraceptives.

Deficient Non-deficient Ever pregnant (n=54) Never Pregnant (n=47) Ever pregnant (n=79) Never pregnant (n=42) Women with VTE, n 17 12 2 3 Observation period, yr 1241 405 2169 430 AI, % (95% CI) 1.37 (0.80–2.19) 2.96 (1.53–5.18) 0.09 (0.01–0.33) 0.70 (0.14–2.04) RR (95% CI) 0.5 (0.2–0.99) P=0.05 0.1 (0.02–0.9) P=0.04 Pregnancy related VTE, n (%) 12 (71) 0 1 (50) 0 Oral contraceptive related VTE, n (%) 2 (12) 9 (75) 0 3 (100)
Risk of Pregnancy-Associated Venous Thromboembolism in Women with a History of Prior Thrombosis.
Authors: Gerhardt Andrea , Scharf Rudiger E. , Zotz Rainer B.
Published: October 2019

Background: Previous estimates of the rate of recurrent venous thromboembolism (VTE) during pregnancy in women with a history of VTE have vary between 0 and 13%. Therefore, the decision to administer or withhold heparin - especially in the antepartum period - has been discussed controversial. In a recent study by Brill-Edwards et al. (N Engl J Med2000;343:1439–44), no recurrences of VTE occurred in women (n=44) who had a previous episode of thrombosis that was associated with a temporary risk factor and who also had no evidence of thrombophilia. Based on these results, antepartum heparin prophylaxis is not routinely recommended in women without thrombophilia whose previous episode of thrombosis was associated with a temporary risk factor (ACCP guidelines 2004). The objective of our study was to evaluate the risk of recurrent pregnancy-associated thrombosis in women with a history of VTE.

Materials and Methods: We retrospectively studied 198 women with at least one pregnancy (275 pregnancies in total) after a one previous episode of VTE. Sixty-three women (81 pregnancies) were excluded from the analysis because of antepartum heparin prophylaxis.

Results: In the subgroup of women without heparin prophylaxis (n=135), 15 (7.7%) thromboembolic events occurred antepartum in 194 pregnancies. Further subgroup analysis, stratified for the nature of first VTE, gave the following number of antepartum VTE per number of pregnancies: 2 VTE/19 pregnancies (10.5%) in 14 women (first VTE: immobilization), 4 VTE/33 pregnancies (12.1%) in 24 women (first VTE: surgery), 5 VTE/69 pregnancies (7.2%) in 46 women (first VTE: oral contraception), 2 VTE/58 pregnancies (3.4%) in 40 women (first VTE: pregnancy), 2 VTE/15 pregnancies (13%) in 11 women (first VTE: idiopathic). Nine of the 15 women with VTE (7/13 women with first VTE triggered by temporary risk factor; 2/2 women with first idiopathic VTE) had a heterozygous factor V Leiden G1691A or prothrombin G20210A gene mutation. In the postpartum period, 16 VTE in 194 pregnancies occurred after live birth in the 135 women without heparin prophylaxis. Nine of these 16 women had a heterozygous FVL or prothrombin G20210A gene mutation. In Conclusion, the risk of recurrent antepartum VTE was similar in women with and without factor V Leiden G1691A or the prothrombin G20210A gene mutation and did not differ between women with first VTE triggered by a transient risk factor or an idiopathic first VTE. In addition to recommended postpartum heparin prophylaxis, our data support the need for a routine antepartum prophylaxis in women without thrombophilia whose previous episode of thrombosis was associated with a temporary risk factor.
Venous Thromboembolism - A Manifestation of the Metabolic Syndrome?.
Authors: Ay Cihan , Tengler Theres , Vormittag Rainer , Simanek Ralph , Dorda Wolfgang , Vukovich Thomas , Pabinger Ingrid
Published: October 2019

There is accumulating evidence for an association between atherosclerosis and venous thrombosis, which may share common risk factors. The metabolic syndrome (MetSyn), a cluster of interrelated risk factors comprising abdominal obesity, elevated blood pressure, high triglycerides, reduced high-density lipoprotein cholesterol and elevated fasting glucose plasma levels is associated with atherosclerotic disease and type 2 diabetes mellitus. It induces a proinflammatory and prothrombotic state. Despite its high prevalence, data on the association with venous thromboembolism (VTE) are scarce. Therefore, we aimed to investigate the association of the MetSyn with the risk for VTE and conducted a case-control study to evaluate the prevalence of the MetSyn according to guidelines of the National Cholesterol Education Program in a high-risk population of patients with objectively confirmed recurrent VTE, who had at least one unprovoked event of deep venous thrombosis or pulmonary embolism. Age and sex-matched healthy individuals served as controls. Finally, a total of 116 patients (53 female, mean age +/−SD: 56 years +/−12) and 129 controls (66 female, mean age +/−SD: 53 years +/−11) were recruited between January 2005 and November 2005. The prevalence of the MetSyn was statistically significantly higher in patients (40/116, 35%) than in controls (26/129, 20%, p=0.012). The unadjusted odds ratio (OR) of the MetSyn for VTE was 2.1 (95% CI [1.2–3.7], p=0.012) and remained statistically significant after adjustment for factor V Leiden, prothrombin G20210A variation and elevated factor VIII activity, sex and age (OR=2.2, 95% CI [1.1–4.2], p=0.024). Furthermore, individuals with the MetSyn (n=66) had significantly higher hs-CRP (median, [interquartile range]: 0.312 mg/dL, [0.142–0.751] vs. 0.153 mg/dL, [0.073–0.330], p<0.001), fibrinogen (390 mg/dL, [342–432] vs. 343 mg/dL, [310–394], p<0.001) and factor VIII activity (182%, [157–216] vs. 159%, [133–199], p=0.005) compared to those without (n=179). In conclusion, the MetSyn was statistically significantly overrepresented in patients with VTE compared to control subjects without a history of venous or arterial thrombosis. Our data suggest that the MetSyn may contribute to the development of VTE as it was associated with a 2-fold increased risk for VTE.
Thiopurine methyltransferase Genotype Is Not a Risk Factor for Secondary Malignant Neoplasias after Treatment for Childhood Acute Lymphoblastic Leukemia on Berlin-Frankfurt-Muenster Protocols.
Authors: Stanulla Martin , Schaeffeler Elke , Moericke Anja , Cario Gunnar , Eichelbaum Michel , Riehm Hansjoerg , Welte Karl , Schrappe Martin , Schwab Matthias
Published: October 2019

The thiopurines mercaptopurine and thioguanine are important components of contemporary polychemotherapeutic treatment protocols for acute leukemias. Thiopurines are prodrugs that undergo intestinal and hepatic metabolism. Activation occurs via a multistep pathway to form thioguanine nucleotides, which are thought to be the major cytotoxic compound through triggering cell cycle arrest and apoptosis. This process is in competition with direct inactivation of thiopurines or their metabolites by thiopurine S-methyltransferase (TPMT). TPMT is a cytosolic enzyme that is ubiquitously expressed in the human body and catalyzes the S-methylation of thiopurines. The TPMT locus is subject to genetic polymorphism, with heterozygous individuals (5 to 11% of Caucasians) having intermediate TPMT activity, and homozygous individuals (0.3 to 0.5% of Caucasians) having low TPMT activity. At least 20 variant TPMT alleles (*2 to *18) have been described so far that confer decreased enzyme activities compared to the TPMT*1 wild-type allele. TPMT genotype is highly concordant with TPMT phenotype. With regard to long-term adverse effects, patients who have diminished TPMT activity were shown to be at increased risk of developing chemotherapy-induced acute myeloid leukemia and radiation-induced second brain tumors tumors after exposure towards mercaptopurine during therapy for childhood acute lymphoblastic leukemia (ALL). To investigate if such an association is generalizable to other entities of secondary malignant neoplasms (SMN) and different treatment approaches for ALL, we collected specimens of 72 patients who developed a SMN after ALL treatment on Berlin-Frankfurt-Muenster (BFM) protocols, analyzed their TPMT genotype and compared genotype frequencies to these in the general ALL patient population. The 72 patient cohort consisted of 49 hematological SMN (half of them received cranial irradiation), 12 brain tumors, and 11 other solid SMN. Neither in the entire patient group nor in subgroup analyses, differences in allele frequencies of TPMT variants conferring diminished enzyme activity were detectable when comparing SMN patients to the overall ALL patient population. Thus, low TPMT activity does not seem to play a major role in the etiology of SMN after treatment for childhood ALL according to BFM treatment strategies. Factors potentially helpful for the explanation of the previously described relationsship of TPMT activity with SMN will be presented.
Identification and Targeting of a PAR1-PAR4 Complex in Human Platelets and Thrombosis.
Authors: Leger Andrew J. , Covic Lidija , Kuliopulos Athan
Published: October 2019

Thrombin is the most potent activator of platelets and plays a crucial role in the development of arterial thrombosis. Human platelets express dual thrombin receptors, a high-affinity PAR1 and low-affinity PAR4. Platelet aggregation studies demonstrated that PAR4 activity is markedly enhanced by thrombin-PAR1 interactions. PAR1-PAR4 co-factoring was shown by acceleration of thrombin cleavage and signaling of PAR4 upon co-expression with PAR1. A combination of bivalirudin (hirulog) plus a novel PAR4 pepducin antagonist, P4pal-i1, effectively inhibited aggregation of human platelets to even high concentrations of thrombin and prevented occlusion of carotid arteries in guinea pigs. Likewise, combined inhibition of PAR1 and PAR4 with small molecule antagonists and pepducins was effective against carotid artery occlusion. Co-immunoprecipitation and fluorescence resonance energy transfer studies demonstrated that PAR1 and PAR4 associate as a hetero-oligomer in human platelets and recombinant fibroblasts. Together these studies show that PAR1 and PAR4 form a stable hetero-oligomer that enables thrombin to act as a bivalent functional agonist. These studies suggest that targeting the PAR1-PAR4 hetero-oligomer may present a novel therapeutic opportunity in the prevention of arterial thrombosis.
Platelets Lacking Sialic Acid Clear Rapidly from the Circulation Due to Ingestion by Asialoglycoprotein Receptor-Expressing Liver Macrophages and Hepatocytes.
Authors: Louise Soerensen Anne , Wandall Hans H. , Patel Sunita , Richardson Jennifer , Italiano Joseph , Clausen Henrik , Stossel Thomas P. , Hartwig John H. , Hoffmeister Karin M.
Published: October 2019

Although surface sialic acid is considered a key determinant for the survival of circulating blood cells and glycoproteins, its role in platelet survival is unclear. We investigated the importance of sialic acid for platelet clearance using mice deficient in the ST3GalIV sialyltransferase gene (ST3GalIV−/− mice) and identified a novel clearance mechanism previously unrecognized for platelets. ST3GalIV catalyzes the addition of sialic acid onto exposed galactose residues of cell surface glycoproteins. ST3GalIV−/− mice have increased platelet surface galactose exposure, a 70% reduction in platelet count, and prolonged bleeding times. We report that ST3GalIV−/− platelets transfused into wild-type C57BL/6J mice exhibit markedly reduced recoveries and shortened survivals respectively compared to littermate wild-type platelets. Infusion of asialofetuin, an antagonist of the asialoglycoprotein-receptor (ASGPR) restored platelet recovery time and initial circulation of ST3GalIV−/− platelets to normal values. Immunohistochemical studies of organ specimens harvested shortly after transfusion of biotin-labelled platelets demonstrated the predominant clearance of ST3GalIV−/− platelets by the liver Kupffer cells and, unexpectedly, hepatocytes. Megakaryocytes cultured from ST3GalIV−/− mice produced proplatelets normally compared to megakaryocytes generated from wild-type littermates, indicating that the thrombocytopenia in the ST3GalIV−/− mice is not due to reduced platelet production. Comparison of ST3GalIV+/+ and ST3GalIV−/− platelet surface receptor expression as evidenced by flow cytometry and preliminary in vitro activation studies did not reveal any significant differences in the two genotypes. We conclude that the absence of terminal sialic acid residues on platelet surfaces exposes galactose residues to the lectin domain of ASGPR on both hepatocytes and liver Kupffer cells, resulting in platelet clearance from the circulation.
Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain.
Authors: Stojanovic Aleksandra , Flevaris Panagiotis , Xi Xiaodong , Chisti Athar , Phillips David , Lam Stephen C.-T. , Du Xiaoping
Published: October 2019

Outside-in signaling of β3 integrins induces and requires phosphorylation at tyrosine-747 (Y747) and tyrosine-759 (Y759) of the β3 subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of β3 cytoplasmic domain. Here we show that tyrosine phosphorylation in the synthetic β3 cytoplasmic domain peptide inhibits calpain cleavage. In platelets, tyrosine phosphates inhibitor, sodium vanadate, enhances thrombin-induced phosphorylation at Y747 and Y759, which is associated with the reduced integrin cleavage by calpain. The effects of sodium vanadate is unlikely to be caused by its effects on calpain activity but is likely to be caused by the susceptibility of integrin cytoplasmic domain, because sodium vanadate did not affect the calpain cleavage of another substrate, fodrin, in platelets. To further support the protective effect of tyrosine phosphorylation against calpain cleavage, we show that mouse β3 (DiYF) with both Y759 and Y747 mutated to phenylalanine is more susceptible to calpain cleavage than wild type during thrombin-induced platelet aggregation. Furthermore, phosphorylation at Y747 and Y759 of β3 in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Y759 is associated with calpain cleavage at Y759. Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the β3 cytoplasmic domain.
Whole Liver Iron Overload Measurement by Magnetic Iron Detector (MID). A Non Cryogenic Bio-Susceptometer.
Authors: Marinelli Mauro , Beruto Piergiorgio , Gianesin Barbara , Lavagetto Antonella , Lamagna Martina , Oliveri Eraldo , Saccone Maria Luigia , Sobrero Giuliano , Terenzani Laura , Luca Gian
Published: October 2019

Accurate assessment of body-iron accumulation is essential for diagnosis and therapy of iron-overload in diseases such as thalassemia, hereditary hemochromatosis and other forms of severe congenital or acquired anemias. At present, the gold standard to determine liver-iron concentration (LIC) is the invasive liver needle biopsy. This technique might lead to large error, in assessing iron burden, due to the heterogeneous distribution of iron deposition in the liver. SQUID bio-susceptometer and MRI are currently the only non-invasive validated methods for LIC measurements. The susceptometer presented herein, named Magnetic Iron Detector (MID), measures directly the iron overload in the whole liver. All of its components operate at room temperature. Since February 2005 about 150 patients and 90 healthy volunteers have been measured and the measures were obtained in blind. The local Ethics Committee approved the study and all subjects gave informed consent. The result of correlations with the LIC measurements by SQUID susceptometry (Dr. A. Piga, Turin) in 43 patients showed a R 0.86 (Fig 1). In 2 patients, affected by Congenital Hemocromatosis, we correlated the LIC measurement by MID with the assessment of the expected iron depletion obtained with the phlebotomy therapy R 0.94 (Fig 2). All the measurements were correlated with the serum-ferritin concentration values R 0.72. We obtained correlation with the LIC measurement by liver biopsy in 7 patients R 0.89, further measures are in progress. The reproducibility of the iron overload of the same patients, measured after a relatively short lapse of time, is better than 0.5g. In conclusion the data obtained shows that MID is a reliable instrument for the diagnosis of the liver iron overload and for the follow-up of the chelation therapy. It is simpler to operate being manageable directly in the Clinical Center and more affordable than competing techniques.

Fig. 1 LIC measured by MID vs LIC measured by SQUID Fig. 1. LIC measured by MID vs LIC measured by SQUID Fig. 2 The iron reduction of two hemochromatosis patients, under phebotomy therapy, compare with the reduction measured by the MID. Fig. 2. The iron reduction of two hemochromatosis patients, under phebotomy therapy, compare with the reduction measured by the MID.
Thyroid Function in Thalassemic Patients Treated with Combined Chelation Therapy.
Authors: Farmaki Kalistheni , Angelopoulos Nicholas , Anagnostopoulos George , Goula Anastasia , Pappa Christina
Published: October 2019

Recently introduced chelation regimens that combine deferoxamine (DFO) and deferiprone (DFP) have been shown to have greater efficacy in promoting iron excretion than either chelator alone and have been associated with rapid reduction of the iron load in the heart and liver, and with reversal of cardiac dysfunction. It is unclear whether this combined therapy could be associated with a decline in the severity of iron-induced endocrinopathies. The primary endpoint of the present study was to investigate the effects of this therapy on the thyroid function in thalassemic patients with subclinical hypothyroidism (SH) or normal thyroid function. Starting in January 2001, 42 patients with b-tlalassaemia major, previously maintained on subcutaneous DFO only, were switched to combined treatment with DFO and DFP. Before the initiation of combined therapy 14 patients had overt hypothyroidism and were treated with thyroxin substitution. The thyroid function of the remaining patients with normal fasting levels of both FT4 and FT3 was further assessed with TRH test. TSH was measured at 0, 30, 60 and 90 minutes after IV injection of 200 mcg of TRH. 15 patients with normal TSH responses (7 males, 8 females, age 28.86 ± 2.20 years, mean ± SEM), and 13 patients (6 males, 7 females, age 31.15 ± 1.85 years), who were considered suffering from SH were finally enrolled. Criteria for the diagnosis of SH was an elevated basal TSH concentration (>5 TSH μIU/ml) or an increment of the TSH levels during the test more than 20 μIU/ml from the basal value. Combination therapy markedly decreased ferritin levels (585 ± 457 vs. 2124 ± 456 μg/l, P < 0.001 in SH group and 868 ± 339 vs. 2877 ± 552 μg/l, P < 0.001 in eythyroidal group). At the time of reassessment (June 2006), the levels of TSH were decreased at all times during the second TRH test in patients with SH: Basal TSH: 4.12 ± 0.63 vs. 6.27 ± 1.08, P=0.01. TSH At 30′ mins: 22.13 ± 2.18 vs. 34.06 ± 4.75, P=0.005. TSH At 60′ mins: 15.89 ± 1.13 vs. 25.69 ± 3.72, P=0.002. TSH At 90′ mins: 11.83±1.26 vs. 19.44±3.27, P=0.001. TSH quantitative secretion, calculated as the area under the curve, was also significantly decreased with combined therapy (1380±118) compared with the initial assessment (2178±312, P=0.004), while no change occurred in basal FT4 (1.06 ± 0.04 vs. 1.15 ± 0.08 in 2001, normal range 0.71–1.85 ng/ml) and FT3 levels (1.55± 0.07 vs. 1.59± 0.08 in 2001, normal range 1.45–3.48 pg/ml). Nevertheless, 7 patients with previous SH exhibited normal results in the reassessment. In patients with previous normal thyroid function TSH response to TRH stimulus was significantly improved only at 60′ mins (9.78±0.73 vs. 11.49±1.28, P=0.03). This study showed that the combination of DFO and DFP followed by an intensive iron chelation might be associated with an improvement in thyroid function in the early stages of hypothyroidism.
Mac-1 and F4/80 Surface Markers Are Present on Murine Hematopoietic Stem Cells.
Authors: Zomorodian Toska J. , Greer Debbie , Wood Kyle , Foster Bethany , Demers Delia , Johnson Kevin , Quesenberry Peter J. , Abedi Mehrdad
Published: October 2019

Transplanted bone marrow donor cells with tissue specific phenotypes have been found in the brain, liver, heart, skin, lung, kidney, and gut of transplanted humans and mice. Such observations have led to the controversial hypothesis that hematopoietic stem cells (HSC) might be intrinsically plastic, and through transdifferentiation or fusion lead to the repair of damaged tissues throughout the body. Alternately, it is suggested that fusion of macrophages to the recipient cells may explain this phenomenon. We have shown recently that purified HSC are the cells responsible for GFP positive donor-derived muscle fibers in the recipient mice post bone marrow transplantation. However, further studies sorting for macrophage markers Mac-1 and F4/80 also resulted in donor-derived muscle fibers in the host. To address this discrepancy, we investigated subpopulations of Mac-1 and F4/80 positive cells, in the presence or absence of stem cell markers (Sca-1 and C-kit). We demonstrate that only the subpopulations of Mac-1 and F4/80 positive cells harboring stem cell markers, Sca-1 or c-kit, were capable of contributing to the regenerating muscle post transplantation. Furthermore, these same subpopulations demonstrated single cell High Proliferative Potential (HPP) (6–26%) in a 7 factor cytokine cocktail, compared to the Mac-1 or F4/80 cells with no stem cell markers (0%). Additionally, they demonstrated long-term engraftment in all three lineages at 1-year (average chimerism of 55% versus 0% in stem cell marker negative groups). These subpopulations were also evaluated for morphology using Hematoxylin/Eosin (H/E), Wright-Giemsa, and Nonspecific Esterase staining. In the Mac-1 and F4/80 positive groups, those negative for stem cell markers resembled differentiated cells of the myeloid origin (macrophages, granulocytes), while those with positive stem cell markers demonstrated stem cell characteristics. We did not observe any engraftability, donor-derived muscle fibers, or HPP potential for CD14 or cfms positive cells coexpressing stem cell markers, indicating that these markers are more appropriate for identifying macrophages. In conclusion, our studies demonstrate that both Mac-1 and F4/80 surface markers are present on HSC and therefore caution must be taken in the interpretation of data using these macrophage markers. It is reasonable to believe that the use of Mac-1 and/or F4/80 surface markers in a lineage depletion process may result in the loss of a subpopulation of stem cells, and other markers such as CD14 or c-fms may be more appropriate for eliminating differentiated macrophages.
A Phase 1/2 Study of SKI-606, a Dual Inhibitor of Src and Abl Kinases, in Adult Patients with Philadelphia Chromosome Positive (Ph+) Chronic Myelogenous Leukemia (CML) or Acute Lymphocytic Leukemia (ALL) Relapsed, Refractory or Intolerant of Imatinib.
Authors: Cortes Jorge , Kantarjian Hagop M. , Baccarani Michele , Brummendorf Tim H. , Liu Delong , Ossenkoppele Gert , Volkert Angela D.G. , Hewes Becker , Moore Laurence , Zacharchuk Charles , Gambacorti Carlo
Published: October 2019

SKI-606 is an orally available, dual Src/Abl kinase inhibitor shown to be 200-fold more potent than imatinib as an inhibitor of Bcr-Abl phosphorylation in biochemical assays. BaF3 cell lines and primary cells from pts expressing different imatinib-resistant Bcr-Abl mutant proteins are sensitive to SKI-606 in vitro. Unlike imatinib and dasatinib, SKI-606 exhibits no significant inhibition of c-kit or PDGFR. This differential selectivity may result in clinical benefit by altering the safety profile. In the phase 1 portion of this phase 1/2 study, pts in chronic phase with imatinib relapsed or refractory disease were eligible for treatment with SKI-606 once-daily dosing. 18 pts [median age: 62 yrs (range 27 – 72); 14 male; 4 female; median CML duration: 5.8 yrs (range 0.9 – 11.1); and median time on imatinib (n=16): 3.9 yrs (range 0.8 – 6.5)] have been enrolled in the following dose cohorts (mg/day): 400 (3 pts), 500 (3 pts) and 600 (12 pts), and have been on treatment for 30 to 192 days. 17/18 pts remain on study; 1 pt discontinued with disease progression. The following SKI-606-related AEs have been reported (n=15, G1/2): diarrhea (87%), nausea (33%), vomiting (20%), abdominal pain (13%), rash (13%), asthenia (13%), and increased AST/ALT levels (7%). 2 pts treated at 600 mg experienced a G3 toxicity: rash and thrombocytopenia. 5 pts (4 pts at 600 mg and 1 pt at 500 mg) had dose reductions for rash, thrombocytopenia, diarrhea, fever and increased AST/ALT levels. No pleural effusion or pulmonary edema has been reported. Of the 7 pts who entered the study in hematologic relapse and have completed 1 month of treatment, all have achieved complete hematologic response. Of the 7 pts on treatment ≥ 12 weeks (time of first cytogenetic assessment), 3 pts have achieved complete cytogenetic response and 1 pt a minimal cytogenetic response. 6/7 pts who have achieved complete hematologic response had pre-treatment imatinib-resistant Bcr-Abl mutations: M351T; F359V; T315I; F359(V,F); and 2 pts with multiple mutations [L248(L,V) and H396(H,R); H396(H,P) and E286(E,G) and M351(T,M)]. The 3 pts with complete cytogenetic response had mutations: M351T; M244V; and H396(H,P), E286(E,G) and M351(T,M). Based on the emergence of 1 DLT of G3 rash, and additional G2 GI and dermatologic toxicities observed at 600 mg, 500 mg has been selected as the dose for the phase 2 portion of the study. Patients in all phases of CML and Ph+ ALL are now being enrolled. SKI-606 is well tolerated in pts with CML, with a primarily GI and dermatologic safety profile, and with encouraging evidence of clinical activity in imatinib-resistant patients with complete hematologic and cytogenetic responses.
Hematopoietic and Endothelial Progenitor Cells from Mouse Adult Testis Derived Stem Cell Lines.
Authors: Schreiter Jessica , Guan Kaomei , Nayernia Karim , Chapuy Bjoern , Truemper Lorenz , Hasenfuss Gerd , Engel Wolfgang , Wulf Gerald G.
Published: October 2019

Adult mouse testis was recently found to harbour multipotent adult germline stem cells (maGSC) with the differentiation capacity along several mesodermal and epithelial lineages. We here addressed the hematopoietic potential from three multipotent germline stem cell lines in vitro. In the undifferentiated state, these cells expressed stem cell markers including Sca1, Thy1 and c-kit, but lacked markers of definitive hematopoiesis such as CD45. On exposure to in vitro culture conditions favouring hematopoietic differentiation, these cells gave rise to both primitive erythropoiesis with nucleated red blood cells expressing ζ-globin, and definitive hematopoiesis, forming β-globin positive erythroid and myeloid colonies in colony assays. On OP9 stroma cells, the maGSC lines formed mesoderm-like colonies, from which progenitor cell populations for either endothelial or hematopoietic differentiation were isolated. The differentiation patterns and efficiencies were similar to the results obtained from the mouse embryonic stem cell line MPI2. In summary, adult germline derived stem cell lines can be induced to recapitulate early embryonic hemangioblastic development and may thus serve as tools for studies on hematopoietic and angioblastic development as well as applications in regenerative medicine.
Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.
Authors: Newell Jennifer , Fay Philip J.
Published: October 2019

Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.
The Effect of an Arg/Trp Polymorphism in α2-Antiplasmin on Fibrinolysis.
Authors: Christiansen Victoria J. , Jackson Kenneth W. , Lee Kyung N. , Chissoe Dini , McKee Patrick A.
Published: October 2019

α2-antiplasmin (α2AP) is a serine proteinase inhibitor with plasmin as its primary target. Plasmin plays a critical role in fibrin proteolysis and tissue remodeling and is highly regulated to prevent excessive proteolysis. α2AP is cleaved by antiplasmin-cleaving enzyme (APCE) between Pro12-Asn13 to yield a shortened version with Asn as the amino-terminus. Asn-α2AP is crosslinked into fibrin ~13 X faster than the full-length Met-α2AP. Clot lysis is slowed in direct proportion to the ratio of Asn-α2AP to Met-α2AP present during fibrin formation. The cloned gene for Met-α2AP contained either a CGG codon resulting in Arg as the sixth amino acid or TGG, resulting in Trp at that position. Both polymorphic forms of Met-α2AP, Arg6 and Trp6, have been found in human plasma. In the present study we determined the genotype frequency of the polymorphism and whether it affected cleavage of Met-α2AP to Asn-α2AP, and ultimately whether fibrinolytic rates were affected by one polymorphic form as opposed to the other. In our normal population, the genotype frequencies were found to be RR 60.9%; RW 35.8%; WW 3.4%. The R allele had a frequency of 78.8% and the W allele had a frequency of 21.2%. A primate DNA panel revealed that the polymorphism follows an evolutionary split with Old World monkeys having the WW genotype and Great Apes having the RR genotype. We found that the polymorphism affected the ratio of Asn-α2AP to Met-α2AP in plasma with WW having the highest percentage of Met-α2AP (56.4%), RR having the least (23.6%) and RW falling in between (40.6%). We examined whether this was due to Met-α2AP(Arg6) being a better substrate for APCE than Met-α2AP(Trp6). When purified α2AP(Arg6) or α2AP(Trp6) were digested with APCE, comparisons of reaction rates based on linear regression analysis of early time points revealed that APCE cleaved Met-α2AP(Arg6) ~8-fold faster that Met-α2AP(Trp6). This phenomenon was confirmed in whole plasma, by incubating plasma from subjects with either the RR, RW or WW genotype and determining the Asn-α2AP/Met-α2AP ratio at 0, 24 and 48 hours. When APCE was removed from the plasma by a specific antibody bound to chromatography beads, the ratio of Asn-α2AP to Met-α2AP did not change over the same time period. Since Asn-α2AP/Met-α2AP ratios are in direct proportion to clot lysis times, we determined whether the polymorphism also impacts and correlates with correspondent plasma clot lysis times. The first tertile of plasma clot lysis times contained all of the WW persons; the longer lysis times in the second and third tertiles contain only RR and RW persons. We conclude that the Arg6Trp polymorphism is functionally significant in that it clearly affects conversion of Met-α2AP to Asn-α2AP, and as a consequence, the rate of Asn-α2AP incorporation into fibrin. Based on these data, the Arg6Trp polymorphism may play a significant role in governing the long-term deposition/removal of intravascular fibrin.
Identification of a Thrombin-Interactive Site within Factor VIII A2 Domain Responsible for the Cleavage at Arg372.
Authors: Nogami Keiji , Shima Midori , Saenko Evgueni L. , Takeyama Masahiro , Tatsumi Kohei , Fujita Yuri , Tanaka Ichiro , Yoshioka Akira
Published: October 2019

Factor VIII is activated by limited proteolytic cleavage at Arg372, Arg740, and Arg1689 by thrombin or factor Xa. Recently, it has been reported that the C2 domain and the acidic region comprising residues 389–394 in the A2 domain interacted with thrombin to facilitate cleavage at Arg1689 and Arg740, respectively. However, a thrombin-interactive site(s) responsible for cleavage at Arg372 remain to be determined. In the current study, several approaches were employed to examine the thrombin-interactive site(s). Thrombin-catalyzed factor VIII activation was inhibited by the addition of one anti-A2 monoclonal antibody 413 with an epitope of residues 484–509 in a dose-dependent manner using one-stage clotting assay. SDS-PAGE analysis revealed that cleavage rate at Arg372 in the heavy chain by thrombin selectively decreased by ~25% in the presence of the antibody. However, cleavage at Arg740 was not affected. Direct binding experiments by both surface plasmon resonance-based assay and ELISA using anhydro-thrombin (Ah-thrombin), catalytically inactive derivative of thrombin in which the active-site serine was converted to dehydroalanine, showed that anti-A2 antibody 413 inhibited the A2 subunit binding to Ah-thrombin. A synthetic peptide corresponding to the 484–509 residues also inhibited the A2/Ah-thrombin binding by ~40%. Mutant A2 molecules where the basic residues in the A2 domain were converted to alanine were evaluated for the binding of Ah-thrombin. Among the tested mutants, K466A mutant possessed ~330-fold lower affinity than the wild type A2 (Kd; 18400 nM, 55 nM, respectively), and the affinities of R471A, R484A, R489A, R490A, H497A, K499A, and K523A were decreased by 2–5-fold (Kd; 259, 212, 183, 163, 123, 255, and 165 nM, respectively). These findings demonstrate that clustered basic residues, in particular Lys466, encompassed the 484–509 region play a key role of thrombin-binding site within the A2 domain, responsible for thrombin-catalyzed factor VIII activation by cleavage at Arg372.
Absence of CD81 Paradoxically Results in a Hyper-IgM and IgG Response to T-Independent Antigens.
Authors: Sanyal Mrinmoy , Shoham Tsipi , Fernandez Rosemary , Levy Shoshana
Published: October 2019

The tetraspanin CD81 is required for numerous biological functions including fertilization, infection, cell migration and cellular interactions in the nervous and immune systems. In B cells CD81 is a component of the CD19/CD21 signaling complex. CD81 was shown to facilitate the redistribution of the B cell receptor (BCR) complex and CD21 into lipid rafts in response to co-engagement, and to modulate BCR signaling. In addition, CD81-deficient mice express low levels of cell surface CD19, thereby potentially altering signaling by the CD19/CD21 co-receptor complex. Interestingly, the onset of CD81 expression coincides with the onset of CD19 expression during B cell development. The foregoing observations suggest that CD81 might reduce the in vivo response of B cells to antigenic stimulation. To test this hypothesis we compared the response of CD81-deficient and wild type mice to T-independent (TNP-LPS) and T-dependent (TNP-KLH) antigens. Surprisingly, CD81-deficient mice mounted significantly higher IgM responses against both types of antigens. Moreover, the IgG response of CD81-deficient mice was stronger and persistent in response to T-independent antigen. We further found that CD81-deficient mice have an increase in bone marrow perisinusoidal B cells (IgM+IgD+). These cells are primarily responsible for mounting T-independent immune responses against blood-borne pathogens. In addition, CD81-deficient spleenic B cells have an intrinsic ability to produce higher amounts of IgM. These surprising results suggest that CD81 is involved in modulating B cell activation, particularly in response to infection.
Enhanced IL-6 Production by Bone Marrow Derived Dendritic Cells from Lupus-Prone Mice Inhibits CD4+CD25+ T Cell Mediated Suppression.
Authors: Wan Suigui , Xia Changqing , Morel Laurence
Published: October 2019

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus due to the co-expression of the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of anti-nuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg), and that Sle3 results in intrinsic defects in myeloid cells that hyperactivate T cells. Here, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone-marrow derived DCs, plasmacytoid and myeloid DCs express a significantly lower level of CD80, CD86 and class II MHC than B6 controls. B6.TC DCs also induce a higher level of proliferation in CD4+ T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs over-produce IL-6, which is necessary for the blockade of Treg activity, as shown by anti-IL-6 neutralizing antibody in the suppression assays. The over-production of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg, but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment both in number and function, and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.
Liver Fibrosis Assessment by Ultrasound Elastometry in Thalassemic Patients on Long-Term Iron Chelation.
Authors: Longo Filomena , Marletto Fabio , Rivetti Elisa , Sacchetti Laura , Piga Antonio
Published: October 2019

BACKGROUND

In thalassemic major patients liver fibrosis may be influenced by independent factors as hepatitis, degree of iron loading and iron chelation, and the specific effect of drugs. Due to its invasive nature, the histological evaluation of liver fibrosis on biopsy samples is difficult to be done and repeated. We analysed the relationship of these factors with the degree of liver fibrosis assessed non-invasively by ultrasound elastometry expressed as liver stiffness. We also compared the liver stiffness in patients on long term therapy with different iron chelators.

METHODS

We applied the ultrasound elastometry (Fibroscan®, Fibrosens, Paris) to a consecutive series of patients with ß thalassemia major on regular transfusion and chelation. Liver Iron Concentration (LIC) has been assessed by SQUID magnetic susceptometry (Tristan Technologies, Inc., San Diego, USA). We considered, as control, a group of healthy persons. All the subjects signed a written informed consent.

RESULTS

We studied 115 patients (64 males, 51 females) with beta thalassemia major, with mean age of 27.7 ± 7.6 years (range: 6.7–48.3). Eighty-six (75%) patients were HCV-Ab positive, and 52 (45%) HCV-RNA positive. Mean stiffness was 9.0 ± 6.6 KPa (range 3.3–43.5), significantly higher than in controls (4.0 ± 1.0, CI 3.3–4.8). Seven (6%) patients had a stiffness value above the cirrhosis threshold, and 18 (16%) were above the severe fibrosis threshold. Mean serum ferritin was 2064 ± 2011 μg/L, and mean LIC was 1845 ± 1199 microg/g ww. Considering the chelator, 34 patients have been on deferoxamine (DFO) therapy for 19.2 ± 9 years, 53 patients on deferiprone (L1) for 5.7 ± 3.7 years, and 28 on deferasirox (ICL670) for 3.3 ± 1.1 years. Mean stiffness was 9.5 ± 7.4 kPa, 9.9 ± 7.3 and 6.6 ± 2.8, respectively. On multivariate analysis, HCV Ab, HCV RNA and LIC were independent factors determining the degree of stiffness, while the chelator used was not significant.

CONCLUSIONS

Thalassemia major patients on regular transfusion and chelation have significantly abnormal degree of liver fibrosis. HCV positivity and the amount of iron overload seem to be the main related factors. The type of chelator used does not seem important.
Detachment of Human Endothelial Cells from Fibrinogen Induced by Cleaved High Molecular Weight Kininogen (HKa) Is Mediated by c-Jun Amino-Terminal Kinase (JNK) Signaling Pathway.
Authors: Liu Yuchuan , Sainz Irma M. , Pixley Robin A. , Colman Robert W.
Published: October 2019

We have shown that HKa inhibits angiogenesis by inducing detachment and apoptosis of human umbilical vein endothelial cells (HUVECs) from specific extracellular matrix proteins, such as vitronectin and fibrinogen, but not collagen. To investigate which intracellular signaling pathway led to cell detachment, several specific inhibitors were selected and utilized. SP600125, a JNK inhibitor, and PP2, a Src kinase inhibitor, both produced HUVECs detachment from surfaces coated with fibrinogen while MG-132, a NF-κB inhibitor, induced apoptosis. Neither U0126, an ERK inhibitor, nor SB202190, a p38 inhibitor, detached HUVECs from fibrinogen surfaces. By Western blot analysis, we found that HKa inhibited JNK, but not Src kinase activity. Previous investigators have demonstrated Rho-associated protein kinase (ROCK) regulates the JNK pathway. We demonstrated that Y27632, a ROCK inhibitor, not only inhibited JNK activity, but also caused detachment of HUVECs. Using phospho-antibodies, we found that HKa decreased the phosphorylation of paxillin at Ser178 site, but not Tyr 118 site indicating a serine kinase. Paxillin is a multi-domain adaptor found at the interface between the plasma membrane and the actin cytoskeleton. Paxillin phosphorylation is known to be associated with the coordinate formation of focal adhesions and stress fibres. Since the Ser178 of paxillin is a substrate for JNK, while Tyr 118 is a substrate for FAK and Src, it appears that JNK is responsible. Our studies have uncovered the novel finding that HKa detached HUVECs from fibrinogen through the ROCK-JNK-paxillin (Ser178) pathway, but not Src-paxillin (Tyr118) signaling.