bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentration of Neurotransmitter and Neurotrophic Factors in Platelet Rich Plasma.
Authors: Swift Matthew J. , Greene Jesse A. , Welch Zachary R. , Woodell-May Jennifer
Published: October 2019

Use of autologous platelet rich plasma (PRP) is emerging in a variety of clinical settings. Although the desired effects of PRP application vary across indications, the common mechanism of action is the up-regulation of the healing response at the site of application. This effect has largely been attributed to the release of cytokines from alpha-granules during platelet degranulation. Research has demonstrated elevated concentrations of a variety of cytokines in PRP samples when compared to baseline blood. To date, no researchthat quantifies the concentration of neurotransmitter and neurotrophic agents present in activated PRP samples has been reported. A number of these molecules have been reported to be present in the alpha and dense granules of platelets with release occurring upon platelet degranulation. Upon release, these molecules elicit actions related to the hemostatic, inflammatory, and reparative processes during the natural wound healing response. In the current study, a commercially available platelet concentrator (GPS II System, Biomet Biologics, Inc) was used to prepare PRP from whole blood samples obtained from 9 healthy subjects. PRP and whole blood samples from each subject were analyzed using a hematology analyzer (Cell Dyn 3700, Abbot Laboratories) for cellular content and then activated with a bovine thrombin/calcium chloride solution. Following a 10-minute incubation, the activated samples were centrifuged for 5 minutes and the resultant serum was collected and assayed for serotonin, adrenaline, noradrenaline, dopamine, and brain derived neurotrophic factor (BDNF) using commercially available ELISA kits. Platelet concentration averaged 196x103 platelets/ml in the baseline samples and 1230 x103 platelets/ml in the PRP samples, a 6.28 fold increase. Serotonin serum levels increased 4.48 fold (base=201.9 pg/ml, PRP=904.6 pg/ml), noradrenaline serum levels increased 3.02 fold (base=286 pg/ml, PRP=863 pg/ml), and BDNF serum levels increased 3.06 fold (base=3.49ng/ml, PRP=10.6 ng/ml). Adrenaline results were highly inconsistent, a factor attributed to the potential of glandular release in response to the blood draw stick. Dopamine concentrations were not detected in any samples using an ELISA with a sensitivity of 100 pg/ml. This study confirms the presence of elevated neurotransmitter and neurotrophic concentrations in activated PRP samples when compared to corresponding base samples. Further studies are necessary to elucidate the role these elevated concentrations contribute in the up-regulation of the wound repair process reported during the clinical application of PRP.
Platelet ABCA1 Expression Is Normal in Scott Syndrome Dogs.
Authors: Catalfamo James L. , Brooks Marjory B. , Ammersbach Melanie , DeMaster Amanda K.
Published: October 2019

Scott syndrome is a rare platelet function defect characterized by a failure of Ca2+-stimulated phosphatidylserine (PS) externalization and lack of platelet prothrombinase activity. In 2005 Albrecht et al. (Blood 106:542) described low levels of ATP-binding transporter A1 (ABCA1) mRNA in lymphocytes and heterozygosity for a novel ABCA1 missense mutation in a patient with Scott Syndrome. The proband’s expression of platelet ABCA1, however, was not measured. Platelet dysfunction has been described as a feature of Tangier disease, a monogenic disorder caused by ABCA1 mutations (Nofer et al, JBC279:34032, 2004). The disease phenotype results from altered lipid trafficking, with a virtual absence of plasma HDL cholesterol and accumulation of lipid in cells of the RE system. In the current study, we systematically evaluated platelets from Scott syndrome dogs to find evidence of ABCA1 deficiency. Platelets from control dogs and affected German shepherd dogs (GSD) were compared with regard to cholesterol content, ABCA1 protein expression, and reactivity to collagen, convulxin (CVX), and thrombin. All GSD had normal plasma cholesterol and triglyceride levels, with no significant difference in mean platelet cholesterol content from that of control dogs (control cholesterol = 13.4 ug/108 platelets, GSD cholesterol = 12.6 ug/108 platelets; n= 6; p = 0.726). Western blot analysis of platelet extracts from control dogs and GSD using a polyclonal antibody to human ABCA1 revealed comparable levels of a 220 kD protein band, the expected size for human ABCA1. We found no significant differences in maximal platelet aggregation in response to 12 ug/mL collagen stimulation [control dog mean response = 82.9% (n=10), GSD = 75.0 % (n= 7); p = 0.133)]. CVX concentration-response aggregation curves obtained for washed platelets were similar for control dogs (n=4) and GSD (n=3) in the range of 1 to 100 nM CVX, with a plateau of maximal aggregation at 10 nM CVX. In flow cytometry analyses, we found no difference in mean platelet mepacrine uptake between control dogs and GSD (mean positive platelets = 64%, n= 3 control, n=5 GSD). There were no significant differences in mean CD62P expression post-thrombin (0.5 U/mL) or CVX (2 nM) stimulation [control CD62P positive platelets post-thrombin = 57%, post-CVX = 47.5% (n=7); GSD CD62P positive platelets post-thrombin = 49%, post-CVX = 56% (n=4)]. Although ABCA1 has been proposed to play a role in PS transport, we found no evidence of ABCA1 deficiency and none of the characteristic platelet abnormalities associated with Tangier disease in canine Scott syndrome, a model of impaired transmembrane PS movement.

Comparison of Platelet Phenotypes ANALYSIS TANGIER DISEASE SCOTT SYNDROME GSD Ionophore induced PS expression Present Absent Collagen or CVX induced aggregation Decreased Normal CVX or thrombin induced alpha granule release Decreased Normal Platelet ABCA1 expression Absent Present
Deficiency in TDAG51 Decreases Atherosclerotic Lesion Development in ApoE-Deficient Mice.
Authors: Hossain Gazi S. , Zhou Ji , Maclean Kenneth , Lhotak Sarka , Sood Sudesh K. , Patel Hansa , Capone John , Rho J. , Austin Richard C.
Published: October 2019

T-cell death associated gene 51 (TDAG51) is a pro-apoptotic gene that can be induced by endoplasmic reticulum (ER) stress agents, including homocysteine, tunicamycin, thapsigargin or dithiothreitol. Our previous studies have demonstrated that transient overexpression of TDAG51 elicited significant changes in cell morphology, decreased cell adhesion and promoted detachment-induced programmed cell death (PCD). In support of these in vitro findings, we have further shown that TDAG51 expression was increased and correlated with PCD in the atherosclerotic lesions from apolipoprotein E (apoE)-deficient mice fed hyperhomocysteinemic diets, compared to mice fed control diet. We designed the current study to investigate the effect of TDAG51 deficiency in the development and progression of atherosclerosis. To assess in vivo significance of TDAG51 on atherosclerosis, we have crossed TDAG51-deficient mice with apoE-deficient mice to obtain double knockout mice. Our findings have demonstrated that TDAG51/apoE-deficient mice have a significant decrease in atherosclerotic lesion area, compared to age- and sex-matched apoE-deficient mice. Total plasma cholesterol and triglycerides as well as lipoprotein profiles were similar in both groups. However, TDAG51/apoE-deficient mice presented with increased hepatic steatosis. Further, a significant upregulation of peroxisome proliferator-activated receptor γ (PPAR-γ), a transcription factor required for adipose tissue formation, was demonstrated in TDAG51-deficient mouse embryonic fibroblasts (MEFs), compared to control wildtype MEFs. Interestingly, earlier studies in mice have reported that overexpression of PPAR-γ decreases atherosclerotic lesion development and increases hepatic steatosis - a phenotype similar to that observed in the mouse deficient in both apoE and TDAG51. Collectively, these findings provide evidence that TDAG51 mediates atherosclerotic lesion development and hepatic steatosis through a mechanism involving PPAR-γ.
2006 Prevalence Estimate of Idiopathic Thrombocytopenic Purpura (ITP) in the United States.
Authors: Deitz Anne C. , St. Laurent Samantha A. , Kobayashi Monica G. , Hall Susan A. , Feudjo-Tepie Maurille A.
Published: October 2019

ITP is a rare but serious platelet disorder. It has been associated with fatal hemorrhage and reduced life expectancy (Cohen et al., Arch Intern Med, 2000). The incidence of ITP has been reported in two population-based studies in Europe (Neylon et al., Br J Haem, 2003; Frederiksen and Schmidt, Blood, 1999). However, there are no recent high quality studies that we are aware of regarding the burden of this disease (i.e., prevalence) in the US by gender and age group, and so we undertook this study in IHCIS (Integrated Healthcare Information Services), a US managed care database comprised of over 38 million patients from 30 health plans. ICD-9 code 287.3 (primary thrombocytopenia) was used as a proxy for an ITP diagnosis. Patients were required to have at least one diagnosis code in 2004 and a second diagnosis code in 2003, 2004 or 2005 to help minimize rule-out diagnoses. Continuous enrolment for a defined period of time also was required. Projected 2006 cases were obtained by multiplying the 2004 prevalence proportions by July 1, 2006 US population estimates (http://www.census.gov/ipc/www/usinterimproj). The projected number of patients with ITP in the US in 2006 was estimated to be 85,000 to 108,000, depending on the stringency of the inclusion criteria (i.e., whether the second occurrence of 287.3 in a patient record had to be within six months of the first). Slightly more of these cases were among women (56%) compared to men, and adults were diagnosed more frequently than those aged 0–15. This is the first study to estimate US prevalence counts using a managed care population, which includes both inpatients and outpatients. However, there are several limitations: ICD-9 code 287.3 is not exclusive to ITP. Segal et al. (Am J Hem, 2004) reported that sensitivity and specificity based on a single occurrence of this code was 100% and 89%, respectively, among inpatients, and 84% and 66%, respectively, among outpatients; our study required two code occurrences. Our estimate also assumes that the prevalence rate has remained constant from 2004 to present. Lastly, Frederiksen and Schmidt reported that the incidence of ITP was higher among older individuals. Therefore, we may be under-estimating the true prevalence of this disease in the general population because the elderly are somewhat under-represented in the IHCIS database.
Antibodies Against Platelet Glycoprotein Target Antigens in Antiphospholipid Syndrome (APS).
Authors: Bidot Carlos J. , Jy Wenche , Bidot Carlos , Horstman Lawrence L. , Fontana Vincenzo , Yaniz Miriam , Gonzalez Martha , Ahn Yeon S.
Published: October 2019

Introduction: Antiphospholipid syndrome (APS) is characterized clinically by thrombotic events and the presence of antiphospholipid antibodies (aPLA) and/or lupus anticoagulant (LA). It is frequently associated with thrombocytopenia and anti-platelet antibodies have been implicated by some. However the roles of anti-platelet antibodies in APS have not been elucidated. We previously reported that platelet activation, but not endothelial activation, was associated with thrombosis in aPLA+ patients [Blood, 104:143a, 2004] but the cause of platelet activation was not addressed. In the present study, we investigated the prevalence of anti-platelet antibodies in APS patients, as well as platelet and endothelial activation.

Material and Methods: We evaluated 47 patients with primary APS. Anti-platelet antibodies against GP IIb/IIIa (CD41b), GP Ib/IX (CD 42b) and GP IV (CD36) for IgG and IgM class were measured by PAICA assay [Thromb Haemost76:1820, 1996]. We also measured platelet and endothelial activation markers by flow cytometry: CD62P on platelets, CD31+/CD42+ platelet microparticles (PMP), and CD31+/CD42- endothelial microparticles (EMP).

Results: Of the 47 patients, 34 (72%) were positive for at least one anti-platelet antibody. Looking first at IgG, 18/34 (53%) were positive for GP IV; 17/34 (50%) for GP IIb/IIIa; and 16/34 (47%) for GP Ib/IX. IgM antibodies were 47% (14/30) for GP Ib/IX, 38%(13/34) for GP IIb/IIIa, and 24% (8/33) for GP IV. Platelet and endothelial markers were significantly more common in the anti-platelet antibodies positive group: 40% vs. 21% for CD62P, 40% vs. 28.5% for EMP, and 23% vs. 5% for PMP, respectively. We found that CD62P associated significantly with IgM anti-GP IIb/IIIa (p< 0.05), and PMP with IgM anti-GP IIb/IIIa (p< 0.05), and IgM anti-GP IV (p< 0.05).

Conclusions: Anti-platelet antibodies are common in APS, confirming previous reports. We found that anti-platelet antibodies IgM anti-GP IIb/IIIa, and IgM anti-GP IV were often associated with platelet activation, suggesting that these antibodies may activate platelets to play an important role in the thrombogenesis of APS. These antibodies were also associated with endothelial activation. It remains to be determined which antibodies, APLA and/or anti-platelet antibodies, play a dominant role in the activation of platelet or endothelial cells and contibute most to the pathogenesis of thrombosis in APS.
Platelet and Red Blood Cell Indices in Harris Platelet Syndrome.
Authors: Naina Harris V.K. , Harris Samar
Published: October 2019

Inherited giant platelet disorders are a group of rare disorders characterized by thrombocytopenia, giant platelets and variable bleeding symptoms. Naina et al., described a new giant platelet disorder called Harris Platelet Syndrome (HPS), the most common inherited giant platelet disorder occurring in up to one third of blood donors from north eastern part of Indian subcontinent. HPS is characterized by an autosomal dominant mode of inheritance with normal to severe thrombocytopenia (less than 50x109/L), giant platelets (mean platelet volume more than 10fL) and absent bleeding symptoms with normal platelet aggregation studies. Occasionally abnormalities in red blood cell morphology have been associated with certain giant platelet disorders such as stomatocytosis in Mediterranean Macrothrmboctopenia, dyserythropoiesis in GATA 1 associated macrothrombocytopenia and thalassemia, in X Linked Thrombocytopenia Thalassemia (XLTT).

This study was undertaken to analyze the platelet and red blood cell indices in blood donors with Harris Platelet syndrome. A total of 203 blood donors were included in this study, 101 blood donors from northeaster part of India with MPV more than 12fl (normal 7–10fl) and 102 blood donors from southern part of India. Before blood donation, all donors were questioned about a history of bleeding conditions, in either themselves or their relatives. Blood samples were collected in ethylenediaminetetraacetic acid (EDTA). Automated platelet counts were performed using a Coulter STKS (Coulter, Hialeah, Florida) within 2 hours of collection.

Peripheral blood smears were examined to confirm thrombocytopenia, giant platelets and red blood cell morphology. There was a significant difference between platelet count (Mean ±SD) 136± 40 Vs 262 ± 53 in southern India (p<0.000). Thirty three donors with HPS had a normal platelet count with MPV more than 12fL. MPV was 13.6±0.13 (range 12 to21.9fL) in donors with HPS and 7.3 ±0.6 (range 6–9.2fl) in southern Indian blood donors. The platelet distribution width (PDW) was 17.4±0.8 in donors with HPS and was 16.38±0.5 in southern India blood donors(p<0.000). Though there was a significant difference between hemoglobin, 13.8 ± 1.0 vs and 14.7± 1.1 (P<0.00), there was no significant difference between RDW, MCV, MCH, MCHC. Peripheral blood smear did not show any obvious red blood cell abnormality, but showed giant platelets and thrombocytopenia.

Harris platelet syndrome is associated with normal to severe thrombocytopenia, giant platelets and significant platelet anisocytosis. There was no associated red blood cell abnormalities observed with HPS.
PEGylation of Factor VIII Reduces the Inhibitory Effects of Human Antibody Inhibitors on the Factor VIII Molecule.
Authors: Tang Liang , Pan Clark , Atwal Harpartap , Nixon Jennifer , Barnett Thomas , Murphy John E. , Mei Baisong , Fournel Michael
Published: October 2019

Replacement therapy with plasma-derived or recombinant Factor VIII (FVIII) has successfully reduced mortality and morbidity and improved the quality of life for patients with hemophilia A. However, up to 30% of patients develop antibody inhibitors to the FVIII molecule. Inhibitors reduce the efficacy of FVIII, increase the cost of treatment, and greatly increase the risk for life-threatening bleeding events in these patients. In the hopes of developing a more efficacious FVIII therapeutic for hemophilia A patients with inhibitors, we initiated studies on the PEGylation of B-region deleted FVIII (FVIII-BDD) with different sizes of polyethylene glycol (PEG) and investigated the inhibitory effects of anti-FVIII inhibitors on the activity of PEGylated FVIII-BDDs. Applying a site-specific mutagenesis technique to the FVIII-BDD molecule, an amino-acid residue at the inhibitor binding site was changed to cysteine. Mutated FVIII-BDD was purified and PEG molecules of different sizes were added at the mutation sites through a chemical reaction with the maleimide group on the activated PEG. PEGylated FVIII-BDD molecules were further purified and tested for FVIII activity using the chromogenic assay. To investigate the effects of antibody inhibitors on the PEGylated FVIII-BDD, studies were carried out utilizing inhibitory plasmas collected from 8 hemophilia A patients with confirmed inhibitors and different monoclonal antibodies as controls. Of the 8 patient plasmas tested, 43 kD PEGylated FVIII-BDD was more resistant to antibody inhibitors in 4 patient plasma samples than unmodified FVIII-BDD. In one sample, pre-incubation of FVIII-BDD with inhibitor patient plasma (1:15 dilution) reduced FVIII activity to 5% of the originally activity added, according to the chromogenic assay. By contrast, approximately 20% of original activity was retained for monoPEGylated FVIII-BDD and >40% activity was retained for diPEGylated FVIII-BDD. Overall, the results suggest that PEGylated FVIII-BDDs may retain more FVIII activity in the presence of some FVIII antibody inhibitors compared to FVIII-BDD. It is important to note that there was a positive correlation between the size of PEG added to FVIII-BDD and the amount of FVIII activity retained. This study indicates that the addition of PEG to FVIII molecules through site-specific PEGylation has the potential to increase the resistance of FVIII to the effects of some antibody inhibitors in patients with hemophilia A.
Single-Dose Pharmacokinetics of Murine and Human Recombinant Activated FVII in Mice.
Authors: Steinoe Anne , Karpf Ditte M. , Stennicke Henning R. , Ezban Mirella , Sorensen Brit B.
Published: October 2019

The practical advantages of using the mouse in pharmacokinetic (PK) studies of human recombinant proteins are many and obvious. However, the relevance of studying human proteins in a mouse model is always questionable. The aims of the present study were i) to investigate if the mouse can be used as a model for PK studies of human FVIIa and FVIIa analogues by comparing the clearance of human recombinant FVIIa (hFVIIa) and murine recombinant FVIIa (mFVIIa), and ii) to study the roles of tissue factor (TF) in clearance of FVIIa.

Eighteen NMRI mice were given a single intravenous 1 mg/kg dose of FVIIa or derivative containing trace amounts of iodinated FVIIa or derivative. Eye blood was sampled from anaesthetized mice in the interval of 0–8 h post administration. A sparse sampling schedule was employed in that three blood samples were taken pr. time point and three samples were drawn pr. mouse. The FVIIa plasma concentration was determined from the radioactivity measured by a γ-counter. The PK parameters were calculated by a 2-compartmental method using the WinNonlin software (Pharsight Corporation, USA), and the data was analyzed in one-way ANOVA test.

The pharmacokinetic profile for both proteins was best determined by a 2-compartment analysis. The PK parameters obtained for the two proteins were statistically indistinguishable and showed a rapid distribution phase followed by a slower terminal half-life. The following values were obtained for hFVIIa and mFVIIa: T½ (distribution) was 0.38 h for hFVIIa and 0.71 h for mFVIIa, while a T½ (elimination) of 4.85 h was found for hFVIIa and 5.53 h for mFVIIa. Total clearance values of 34.7 ml/kg/h and 32.8 ml/kg/h were obtained for hFVIIa for mFVIIa, respectively. The volumes of distribution for the two proteins were also statistically indistinguishable, being 220.1 ml/kg for hFVIIa and 203.9 ml/kg for mFVIIa. No significant difference was thus observed between hFVIIa and mFVIIa in terms of the PK parameters including the terminal half-lives. These data might indicate that human FVIIa and murine FVIIa are cleared by the same mechanism(s) in mice, suggesting that the mouse is a valid model for PK studies of human FVIIa and derivatives thereof.

The similar PK profiles for hFVIIa and mFVIIa observed further indicated a TF-independent clearance mechanism of FVIIa. Relative to mFVIIa the affinity of hFVIIa for mTF is known to be markedly decreased (Petersen LC et. al. Thromb Res.2005;116:75–85). Involvement of TF binding in clearance was therefore expected to result in different PK profiles for the two proteins; hence in a mouse model one would expect hFVIIa to be cleared at a much slower rate than mFVIIa. Since this was not the case, this study suggests a TF-independent mechanism of FVIIa clearance in mice.

Our studies suggest that the mice can be used as a model to evaluate the PK profile of human FVIIa, and that TF is not involved in the clearance mechanisms of FVIIa.
Allosteric Disulfide Bonds in Thrombosis and Thrombolysis.
Authors: Hogg Philip J.
Published: October 2019

Allosteric disulfide bonds control protein function by mediating conformational change when they undergo reduction or oxidation. The known allosteric disulfides are characterized by a particular bond geometry, the -RHStaple. A number of thrombosis and thrombolysis proteins contain one or more disulfide bonds of this type. Tissue factor (TF) is the first haemostasis protein shown to be controlled by an allosteric disulfide, the Cys186-Cys209 bond in the membrane-proximal fibronectin type III domain. TF exists in three forms on the cell surface; a cryptic form that is inert, a coagulant form that binds factor VIIa to initiate coagulation, or a signaling form that binds VIIa and cleaves protease activated receptor 2 that functions in inflammation, tumor progression and angiogenesis. Reduction and oxidation of the Cys186-Cys209 bond is central to the transition between the three activities of TF. The redox state of the bond appears to be controlled by protein disulphide isomerase and NO. Plasmin(ogen), vitronectin, glycoprotein 1balpha, integrin beta3 and thrombomodulin also contain -RHStaple disulfides and there is circumstantial evidence that the function of these proteins may involve redox change of these disulfide bonds.
The Effect of Irreversible and Reversible Direct Thrombin Inhibitors and Danaparoid on Tissue Factor Mediated Thrombin Generation.
Authors: Samama Meyer M. , Le Flem Lena , Pierre-Eugène Daphne , Depasse Francois
Published: October 2019

Background

Recently, direct thrombin inhibitors such as Hirudin, Bivalirudin, Melagatran and Argatroban have been introduced in the armamentarium of an antithrombotic drugs. Danaparoid, an anti-Xa - anti IIa agent is also used especially in Heparin induced thrombocytopenia.

Objective

Thrombin generation test (TGT) has been recently automated and could be useful for the evaluation of the inhibitory activity of these drugs and the measurement of their anticoagulant effects at established therapeutic concentrations. Moreover, TGT can elucidate some information about their mechanism of action.

The aim was to compare Hirudin, Argatroban, Melagatran and Danaparoid while Bivalirudin was not available.

Methods and results

Pools of normal human plasma were spiked with increasing concentrations of the four studied drugs and TGT was determined by studying the lag time (LT) and time to peak TTP as well as the peak (P), the endogenous thrombin potential (ETP) and the mean rate of thrombin generation (velocity TG) and its inhibition once a maximal concentration has been reached (velocity TI). The mechanism of action on TGT was heterogeneous. At therapeutic concentrations, Hirudin delays dramatically thrombin generation without affecting significantly its velocity and the amount of thrombin formed.

Argatroban and Melagatran induce a far less marked prolongation of LT and TTP than hirudin but they both reduce the velocity of the reaction and the amount of thrombin generated in this in vitro study. Melagatran was found more active than Argatroban but the patterns of the thrombograms were similar.

Danaparoid exerts a minimal effect on LT and TTP which is associated with a very significant influence on the parameters related to the reaction of thrombin formation (velocity index, peak and ETP). In total ETP generally considered as the most informative parameter of the thrombogram was not found as relevant as predicted. Schematically, 3 different patterns for the thrombogram have been observed indicating different mechanisms of anticoagulation, all of them, clearly associated with an antithrombotic activity in vivo.

Conclusion

The mechanism of action of hirudin on thrombin generation is clearly different from that of Argatroban, Melagatran and Danaparoid. This work demonstrates that antithrombotic activity is associated with different alterations of TGT. The results could help to determine the blood concentrations required for an effective anticoagulation or reciprocally the alterations of the thrombogram which are associated with therapeutic efficacy. Moreover they could be useful when laboratory monitoring of the treatment with these different drugs is considered.
Antiplatelet Agents as Secondary Thromboprophylaxis after Anticoagulant Treatment for Thrombosis.
Authors: Papaioannou Georgia , Papadakis Emmanouil , Topalidou Maria , Kokoviadou Kyriaki , Karapiperidou Anastasia , Kioumi Anna , Korantzis Ioannis
Published: October 2019

Patients suffering an unprovoked VTE event have high risk of recurrence after anticoagulation cessation especially in the first two-year period. Our study examined whether secondary thromboprophylaxis with antiplatelet drugs reduces the relapse risk.

STUDY DESIGN: We studied 53 patients, 25 men and 28 women, (group A), with median age of 41(18–74) years. 31 received aspirin at 100mg while 22 received clopidogrel at 75mg after anticoagulant therapy for VTE. We compared them with 77 patients (group B) who did not receive any thromboprophylaxis after VTE with a median age of 40(18–82) years. Of group B, 56 were men while 21 were women. In 51 out of 130 patients we monitored D-dimers, F1+2 prothrombin fragments, and TAT (thrombin-antithrombin) complexes as prothrombotic and fibrinolysis markers. Failure was determined as VTE recurrence or elevation of prothrombotic markers in the first 2year period.

RESULTS: In 4 patients of group A (7,5%) we noticed marked elevation of prothrombotic markers and they were turned to anticoagulation, and in 3 marker elevation subsided by changing the antiplatelet agent (aspirin to clopidogrel). 1 patient stopped prophylaxis due to hemorrhage while none relapsed. In group B VTE recurrence was noticed at 27(36%, p<0,0002). In both groups late recurrence (> 2 years) was observed at 27,6% of 130 patients.

CONCLUSIONS: Low dose aspirin or clopidogrel administration is safe and effective as thromboprophylaxis after anticoagulation for first venous thromboembolic event. Markers as D-dimers, F1+2, TAT, have prognostic significance and are valuable tools in patient monitoring. Patient follow-up has to be long-standing because VTE recurrence risk persists through the years.
Bcl-2 Family Protein Inhibitors Induce a Unique Thrombocytopenia In Vivo.
Authors: Elmore Steven W. , Bauch Joy , Fryer Ryan M. , Iciek Laurie , Marsh Kennan , Morgan Sherry J. , Nelson Richard , Reinhart Glenn A. , Smith Morey L. , Tse Chris , Rosenberg Saul H.
Published: October 2019

We recently disclosed the discovery of ABT-737, a potent antagonist of antiapoptotic Bcl-2 family proteins that induced tumor regression in murine xenograft models. ABT-263 is a related, orally bioavailable Bcl-2 family protein inhibitor that is currently in clinical development. For both agents, multiple daily dosing from two to four weeks was well tolerated in all species evaluated. The primary preclinical toxicological finding in mouse, rat and dog was a unique thrombocytopenia characterized by a rapid clearance of circulating platelets. A series of in vivo studies that were performed to better characterize this phenomenon are described here. After a single intravenous or oral dose in dogs, circulating platelet counts decreased rapidly and concentration dependently with a nadir at approximately six hours post administration. Platelet counts returned to near normal levels within several days post dose, accompanied by an increase in both mean platelet volume and percent of reticulated platelets. Analysis of platelets by aggregometry during this rebound period indicated that returning platelets were fully functional. Following multiple doses, platelet counts also decreased rapidly after initial dosing, but exhibited evidence of rebound during the dosing period that appeared to be dose dependant. Evaluation of fibrinogen, d-dimer, antithrombin-III, PT and APTT did not support a mechanism involving consumptive coagulopathy. Whole body scintography utilizing [111] Indium labeled platelets in dogs indicated that platelet clearance after compound administration is primarily mediated by the liver. These data suggest that ABT-737 and ABT-263 preferentially affect circulating platelets rather than abrogating platelet production in the bone marrow. In fact, the observed rebound in platelet count in the face of continued dosing reflects the capacity for the animals to compensate for the effect. The enantiomer of ABT-737 (which has significantly lower activity against Bcl-2 family proteins) had no effect on circulating platelets in animals suggesting an underlying mechanism that is dependent on inhibition of antiapoptotic Bcl-2 family proteins. The unusually rapid kinetics of platelet clearance and recovery suggests that the drug-induced thrombocytopenia elicited by these Bcl-2 family protein inhibitors is unique compared to that observed with conventional cytotoxic chemotherapy.
Expression of MTPG-24, a Peptide Derived from a Protein Regulating Dynamic Changes in Cortical Actin, Induces Endomitotic Replication in Megakaryocytic Cells.
Authors: Ste-Marie Alexandre , Simard Carl , Côté Serge
Published: October 2019

Prior to the fragmentation of their cytoplasm leading to platelet formation, differentiating megakaryocytes (MKs) increase their ploidy and cellular volume by undergoing repeated rounds of DNA replication without concomitant cell division, a process known as endomitosis. There is now accumulating evidence that endomitotic MKs develop as a result of aberrant regulation of the cleavage furrow formation, the site where dynamic changes in actin, myosin and membrane structure mediate the process of cytokinesis during animal cell division. Here we report that transfection of human megakaryocytic cell lines with vectors expressing a 24mer-peptide (MTPG-24) resulted in remarkable changes in morphology, resulting in cells with the appearance of mature polyploid megakaryocytes. MTPG-24 is derived from a protein involved in regulating actin-based structures and motility which possesses a putative site for PKA phosphorylation. Despite its hydrophilic primary structure, MTPG-24 fused to a fluorescent tag was detected closely associated to the plasma membrane where it accumulated in a punctuate pattern, in addition to co-localizing with a Golgi marker. Interestingly, in each recombinant clone, cells with different ploidy levels developed among normally dividing ones, suggesting that transit from a mitotic cycle to an endomitotic cycle seemed to only occur in cells having completed a given number of cell cycles. These effects were obtained independently of the nature of the tag fused to the peptide (Hemagglutinin, Orange protein, GFP) but were not observed with a control peptide in which the amino acids of MTPG-24 were disposed in an arbitrarily sequence. Because it possibly interferes with some effector molecules required for cleavage furrow membrane assembly in late anaphase, MTPG-24 provides thus a new tool for understanding the molecular basis of endomitosis. Furthermore, since there is a correlation between DNA content and the numbers of platelets released by MK, we are currently testing whether this peptide could be used to increase the ploidy level of CD34+ human cord blood-derived MKs which have a low propensity to become highly polyploid.
Differential Induction of Apoptosis in Human Leukemia Cells (ML-1, ML-2, CTV-1 and KASUMI-1) Exposed to the Proteasome Inhibitor Bortezomib and the Effect of Transforming Growth Factor-beta Signaling Pathway.
Authors: Fuchs Ota , Provaznikova Dana , Peslova Gabriela , Kocova Marcela , Marinov Iuri , Spicka Ivan
Published: October 2019

Bortezomib (N-pyrazinecarbonyl-L-phenylalanine-L-leucine boronic acid; VELCADE, formely known as PS-341 or MLN-341; Millenium Pharmaceuticals, Inc., Cambridge, MA) is a potent and specific proteasome inhibitor. It is the first member of proteasome inhibitors which obtained approval as chemotherapeutic agent for the treatment of relapsed or refractory multiple myeloma. First clinical experience of bortezomib in patiens with follicular lymphoma and mantle cell non-Hodgkin lymphoma suggest that bortezomib is well tolerated and has significant single-agent activity. Several phase I trials on the use of bortezomib as a novel treatment strategy in leukemia have been started. We studied the effect of of bortezomib and of transforming growth factor-beta (TGF-β) on induction of cell cycle arrest and apoptosis in human leukemia cells (ML-1, ML-2, CTV-1 and KASUMI-1) established from the peripheral blood of patients with acute myeloid leukemia (AML). [6-3H] thymidine incorporation was used as a measure of DNA synthesis to estimate cell proliferation. Apoptosis was detected by flow cytometry using annexin V-FITC/propidium iodide assay and results were confirmed by cell cycle analysis. The profiles of cellular DNA contents indicated the distribution of the cells in different phases of the cell cycle and any possible DNA loss due to DNA fragmentation during apoptosis. The cells with a DNA content less than that of G1 cells were considered as apoptotic cells. Statistical significance of the experimental results was analyzed by Student’s paired t-test. Leukemia cells were preincubated for 24–96 h without addition (control) or with bortezomib (4 nM or 10 nM) or with TGF-β1 (5 ng/ml or 10 ng/ml). TGF-β1 inhibited DNA synthesis only in KASUMI-1 cells but not in other leukemia cells used. Bortezomib (10 nM) was potent inhibitor of DNA synthesis in all four types of leukemia cells and induced apoptosis in KASUMI-1, ML-2 and CTV-1 cells but not in ML-1 cells [Figure 1]. Kinetics of apoptosis was different in individual cell lines. The peak of apoptosis was reached in 24 h in ML-2 cells, however in KASUMI-1 and CTV-1 cells in 48h. KASUMI-1 cells were most sensitive to bortezomib. In addition KASUMI-1 and ML-2 cells were also sensitive to induction of apoptosis by TGF-β1 but in lesser extent than by bortezomib [Figure 1]. Kinetics of apoptosis induction by TGF-β1 was slower than with bortezomib and lasted 48–96 h. Different sensitivity of human leukemic cell lines to bortezomib likely mimics behaviour of primary leukemic cells of patients and thus limits the use of proteasome inhibitors in therapy.

Figure 1 Figure 1.
Experimental Study of the Extract of Hedyotic Diffusa Willd on Induction of the Apoptosis of Leukemia U937 Cells.
Authors: Lin Shengyun , Ye Baodong , Hu Meiwei , Hu Zhiping , Yu Rongxi , Zhou Yuhong
Published: October 2019

Hedyotic Diffusa Willd (HDW) is a Chinese herbal medicine, it has the effect on heat-clearing and detoxicating, activating blood circulation to dissipate blood stasis, removing dampness and promoting diaresis and deturescence. It used for treating various kinds cancer generally for the past few years in china. According the different of solvent polarity, we had extracted the active components from HDW by various solvent (water, ethanol, acetidin, chloroform, alkane) and explored the effects of every active component from HDW on proliferation and apoptosis of leukemia U937 cells. It was found that ethanol extract showed the strongest antitumor effect. Exposured log phase growth U937 cells to different ethanol extract from HDW concentrations for different time respectively and then test the inhibitive effect by modified tertrozalium salt (MTT) assy. The apoptosis of HDW induced on U937 cells was detected by means of optics microscope, flow cytometry and gel electrophoresis of DNA. The antiproliferative effects were observed following exposure ethanol extract of HDW. It shows a very substantial differences in the dose and duration dependency of the observed antiproliferative and cytotoxic effects. The U937 cells treated by ethanol extract of HDW are observed apoptosis characteristic morphological changes such as complete cell membrane,nuclear fragmention apoposic bodies and found trapezoidal belt with DNA.When 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml ethanol extract of HDW treated U937 cells for 48 hours, the numbers of apoptotic cells were respectively 4.18%, 56.96%, 82.43%, 56.41%. These results indicate that Ethanol extract of HDW has the strongs antitumor and inhabited the proliferation and induced the apoptosis of U937 cells stignificantly.
Fluorescence Quantitative PCR Detection of FLT3 Gene Expression in Leukemias and Its Clinical Implications.
Authors: Xu Bing , Tang Jiahong , Li Lin
Published: October 2019

Objective In order to study the clinical significance of the FLT3 expression level in AML patients.

Methods we analyzed the expression level of the FLT3 transcript quantitatively by real time fluorescence quantitative PCR amplification and investigated correlations with FLT3/ITD gene mutation in 69 de novo AML cases.

Results The mean of FLT3 transcripts in the 69 AML patients was 17240 copies/μgRNA(273~834805), which was significantly higher in AML cases than in healthy donors (p=0.017). The FLT3 expressions were not equally distributed among the FAB subtypes(P=0.0005). The leukocyte counts was significantly related to FLT3 expression (P=0.0001 and r=0.4494). There were noticeable deviation among karyotypes in the expression of FLT3. The expression of cases with t(15;17) was lower that that of cases with t(11q23) and complex karyotypes. There was no significant difference of FLT3 mRNA between patiens positive and negtive for CD34 (P=0.315). FLT3/ITD gene mutation was observed in 17 of total 69 cases with AML(24.6%). There was no significant difference of FLT3 mRNAamong FLT3/ITD positive and negtive patients (P=0.205) and there was no significant difference of CR rates in high-FLT3-expression and low-FLT3-expression groups after chemical theropy(P=0. 425). During the follow-up of 210 days(median day=124), there was no significant difference of the cumulative relapse rates between these two groups (P=0.631).

Conclusion The expression of FLT3 gene was significantly higher in patiens with AML than in healthy donors and had a positive correlation with peripheral white blood cells counts. There was statistic difference among various FABs and karyotypes. FLT3 mRNA level in AML patients had no correlation with the expression of CD34 and FLT3/ITD gene mutation. In the short-term follow-up, high expression of FLT3 had no influence on CR, CRR and OS of AML patients.
Classical and Molecular Cytogenetic Abnormalities in 124 Pediatric Patients with Acute Lymphocyte Leukemia.
Authors: Chai Yihuan , Lv Hui , Lu Jun , Xiao Peifang , Li Jianqing
Published: October 2019

In childhood acute lymphocyte leukemia (ALL), cytogenetics plays an important role in diagnosis, allocation of treatment and prognosis. On base of the conventional cytogenetic analysis, molecular methods have inproved our ability to accurately and rapidly risk-stratify patient with childhood ALL in the last few years. Our aim was to assess the demography of cytogenetic abnormalities in childhood ALL. The study sample consisted of 124 newly diagnosed ALL patients younger than 16 years, who were diagnosed at the Department of Pediatric Hematology/Oncology, Soochow University Children’s Hospital. The diagnosis and FAB subtypes of ALL was determined by Wright-Giemsa-stained bone marrow smears and cytochemicalstaining. Immunophenotyping of the bone marrow samples was performed by flow cytometry. Multiplex reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to detect the 29 most common leukemia translocations for routine molecular diagnostic hematopathology practice, and complement the information gained from conventional cytogenetic analysis. Cytogenetic analysis was successful in 112 of 124 children with ALL. Sixty-eight (60%)of them had clonal chromosomal abnormalities. Numerical imbalances consisted of hyperdipoild(>47 chromosomes, 36 cases), hypodipoild(<46 chromosomes, 14 cases), pseudodiploidy(18 cases). Chromosomal translocations were observed in 13 patients by conventional cytogenetic analysis. Three cases were found positive for t (4;11), 3 cases for t (9;22), 1 case for t (1;19) and 6 cases for other rare translocations. RT-PCR analysis detected 116 of the 124 ALL patients. Thirteen cases of TEL-AML1, 10 cases of rearrangement in the MLL gene, 4 cases of E2A-PBX1, 4 cases of E2A-HLF, 3 cases of BCR-ABL, 2 cases of TLS-ERG, 32 cases of HOX11, were detected by RT-PCR in B-lineage leukemias. SIL-TAL1 had been found in 4 of 7 of T-lineage leukemias. Sixty-eight cases of ALL show chromosomal aberrations. Multiplex PCR positivity was detected in 59(50%)of the 116 ALL patients studied. Multiplex PCR combined with chromosome analysis uncovered Chromosomal abnormalities in 95 of 124(77%) of ALL patients and supplemented each other in detecting Chromosomal abnormalities. It provides reliable evidence for the diagnosis, classification and prognosis.
Clinico-Biological Characteristics and Prognosis of Non-M3 Acute Myeloid Leukemia with High Percentage of Myeloperoxisase Positive Blasts. Single Center Experience.
Authors: Montesinos Pau , Algarra Lorenzo , Sanz Jaime , Perez Mari Luz , Senent Leonor , Sempere Amparo , Gomis Federico , Cantero Susana , Martinez Jesus , Lorenzo Ignacio , Sanz Guillermo , Sanz Miguel
Published: October 2019

Introduction: It has been suggested that acute myeloid leukemia (AML) showing mature phenotype is associated with favourable outcome. In a study recently published by JALSG, myeloperoxidase (MPO) positivity in over 50% of blasts had favourable prognostic impact, independent from karyotype, on achieving complete remission (CR), overall survival (OS) and disease free survival (DFS). No other studies have established the independent prognostic value of MPO expression.

Objectives: Analyze the clinico-biological characteristics of AML with high percentage of MPO+ blasts and its impact on CR, OS and DFS.

Material and methods: Between 1986 and 2005, 418 adult patients (median 53 years, range 15–80) were diagnosed with de novo non-APL AML and evaluated for percentage of MPO+ blasts. All patients received intensive chemotherapy. Diagnosis was made by optic microscopy of bone marrow (BM) aspirates stained with May-Grumwald giemsa, MPO, butyrate esterase and or non specific esterase. Cytogenetic and immunophenotype analysis was evaluated in 66% and in 76% of the cases respectively.

Results: 118 patients (28%) showed a percentage of MPO+ blasts >75%. AML with MPO+ blasts >75% was associated with M1-M2-M4 subtypes, leucocytes >50×109/L, blasts in BM >70% and HLA-DR negativity (p<0.01). It was also significantly associated with favourable karyotype (11% vs 3% favourable, 52% vs 48% intermediate and 3% vs 15% unfavourable). Patients with AML and MPO+ blasts >75% obtained higher CR rate (71% vs 55%), due to less resistant disease (9% vs 22%, p<0.01). In multivariate analysis favourable karyotype, leukocytes <50×109/L and age <60 were favourable prognostic factors for CR. Median OS and DFS was higher in patients with AML and MPO+ blasts >75% (15 vs 7 months, p<0.001, y 41 vs 12 months, p<0.001, respectively). ). In multivariate analysis, favourable karyotype, leukocytes <50×109/L, age <60 years and MPO+ >50% were favourable prognostic factors for OS; and age <60 and MPO+ >75% were the only independent factors for DFS. Median DFS was higher in patients with AML and MPO+ blasts >75% in the intermediate cytogenetic risk group (59 vs 13 months, p=0.015), age <60 (109 vs 15 months, p=0.003), age >60 (13 vs 7 months, p=0.03), autologous stem cell transplantation (100 vs 9 months, p=0.04) and chemotherapy alone (16 vs 8 months, p=0.003).

Conclusion: In our series, patients with AML and MPO+ blasts >75% show less chemoresistant disease and a longer remission duration, the latter independently from the karyotype. This biological characteristic could be useful in designing therapeutic strategies in patients that lack other prognostic markers.