bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Analysis of Candidate Modifier Polymorphisms in β-Thalassemia/Hb E Patients.
Authors: Fucharoen Suthat , Winichagoon Pranee , Sripichai Orapan , Munkongdee Thongperm , Kumkhaek Chutima , Vichitumaros Kanjana , Whiacre Johanna , Abel Ken , Braun Andreas
Published: October 2019

β-Thalassemia/Hb E patients encompass a number of clinical severities, ranging from nearly asymptomatic to transfusion-dependent thalassemia major. This has been well documented, but the causes of the variability and the molecular basis of the interaction remain unexplained. In general any factor capable of reducing the degree of alpha-globin-beta-globin chain imbalance will result in a milder form of thalassemia. The major modifying factors demonstrated in the mild cases are coinheritance of mild β+-thalassemia and Hb E genes, coinheritance of α-thalassemia and increased production of Hb F. However, the fact that many patients who have seemingly identical genotypes, β0-thalassemia/Hb E, and do not have a detectable α-thalassemia or increased Hb F production still have mild clinical symptoms, while other patients have a very severe clinical condition similar to homozygous β0-thalassemia. This variation suggests that other unknown modifying genetic factors may contribute to severity of the disease.

To assess the relative contribution of genetic factors in the variation of severity among β-thalassemia/Hb E patients, we conducted a prospective study searching for modifying factors in almost 1100 Thai/Chinese β0-thalassemia/Hb E patients from Thailand using an automated, chip-based platform based on mass spectrometry (Sequenom’s MassARRAYTM system). A map of ~80 single nucleotide polymorphisms (SNPs) has been constructed spanning more than 80 kb, including the locus control region (LCR) and all beta-like globin genes. These SNPs were identified through resequencing and from the public domain, including well-characterized restriction fragment length polymorphisms (RFLPs) used in prior haplotype studies. Included in this panel are assays for polymorphic sites reported to influence globin gene expression, specifically Gγ-Xmn I polymorphism and BP-1 binding site upstream of β-globin gene. Genotyping of other candidate modifier loci, including SNPs in genes encoding alpha hemoglobin stabilizing protein (AHSP), β-globin gene repressor BP-1 protein, erythropoietin (Epo), and transcription factors; GATA-1, EKLF, NF-E2, has been studied. To identify additional modifier loci, carefully selected patient sub-groups representing the extremes in disease severity either mild or severe have been selected for DNA pool construction to be used in a genomewide screen involving up to 100,000 validated gene-based SNPs. It is expected that this genomewide screen will yield important information on the role of candidate genes and may uncover the association of novel polymorphisms with severity heterogeneity in β-thalassemia/Hb E disease.
Transfusion Transmitted Viral Infections in Transfusion Dependent Patients with Thalassemia in Turkey: A Multicenter Study.
Authors: Sonmezoglu Meral , Aydinok Yesim , Canatan Duran , Turker Meral , Gulen Huseyin , Irken Gulersu , Karakas Zeynep , Uysal Zumrut L. , Aydogan Gonul , Cengiz Ozcan , Timur Cetin , Apak Hilmi , Canpolat Cengiz , Bayik Mahmut M.
Published: October 2019

Thalassemia Major (TM) represent one of the largest groups requiring regular transfusions lifelong. A total of 3830 patients are currently registered with homozygous b-Thalassemia in Turkey. This study is designated to reveal the seropositivity status of thalassemia patients on chronic transfusion treatment for blood-borne infections. A questionnaire inquiring the demographics and medical history of the patients as well as serological status for hepatitis B, C and HIV, was sent to 12 Thalassemia Centers which are generally settled down in the University or big government hospitals with high capacity blood banks where the packed red cells were provided. The collected data of 999 patients who have regular transfusions of 1–2 Unit red cells, every 3–4 weeks beginning from the first years of life and screened for transfusion transmitted viruses (TTV) 6 monthly was analyzed. The patients were aged between 0–53 years (median 16 years). Overall HBsAg and Anti-HCV positivity were found 2.5% and 12.6% respectively. Confirmed Anti-HIV positivity was 0.2% in the study group and consistent with the low frequency of the disease among blood donors in Turkey. Age distribution of sero-positivity within the patients was as follows: 0.7% in 0–5 years, 17% in 6–10 years, 48.3% in 11–20 years and 34% over 21 years, indicating the strong relation between the sero-prevalence of TTV and transfused blood volume and the gradually increased safety of blood in Turkey. The highest prevalence rate was seen in the age of 11–20 years which also represents highest number of patients. Donated blood screening for HBsAg, anti-HIV 1/2 (since 1985), anti-HCV (since1996), and RPR for syphilis is obliged by law in Turkey. Although, widespread use of HBV vaccination in transfusion dependent patients has been started since 1990s, the data confirmed that this measurement is insufficient alone for eradicating the HBV transmission in this group. Vaccination against HBV has been scheduled routine vaccination program of infancy by the health authority since 1999. However, the positive reflection of this policy in blood safety would be seen in the next generations. In that point of view, vaccination against hepatitis B can be practised in blood donors as a preventive measure in Turkey which is considered as in the middle endemic region regarding hepatitis B infection. Although, Anti-HCV screening in blood donors caused significant decrease in HCV infection, it still represents the main cause of morbidity following cardiomyopathy in this group of patients. This study clearly shows that the threat of TTV infections among multi-transfused thalassemia patients and underlines the importance of strengthen blood donor selection and implementing the sensitive screening assays for TTV at blood centers.
The Value of Immediate Determination of C-Reactive Protein in the Hematology-Oncology Clinic.
Authors: Shah Vinay M. , Nguyen Hanh T.H. , Ramos Mercedita , Tisman Glenn
Published: October 2019

CRP is named such for its ability to precipitate the somatic C-polysaccharide of Streptococcus pneumoniae. This polypeptide acute phase reactant is a sensitive systemic marker of inflammation, malignancy and tissue damage. ABX Diagnostics developed a hematology analyzer (Micros CRP), which rapidly measures 16 routine hematological parameters plus whole blood CRP. The CRP value, determined by specific antigen-antibody induced optical turbidity, is available within 5 minutes with sensitivity to 0.10 mg/dl. Normal whole blood values range between 0.0 to 0.2 mg/dl. We evaluated the added clinical benefit of immediate CRP determinations in an outpatient cancer clinic in 155 patients undergoing observation and aggressive chemotherapy. Blood was obtained from all patients each visit and routine CBC, Platelet count and CRP were recorded, immediately provided to the physician in the presence of the patient and automatically stored in a database (HemaLink). Correlations were made between changes of CRP, WBC, AGC, and clinical condition. Routine measurement of CRP and its immediate availability for clinical interpretation was found to add a valuable new diagnostic dynamic for cancer patient care. Elevated CRP and its relative change were found to be a leading indicator of serious and frequently subclinical infection and were the earliest clues when compared to WBC or AGC. Relative changes of CRP were found to be indicative of clinical course. Diagnostic value of the WBC in cancer patients was lost in those undergoing myelosuppressive therapy and in those receiving leukocyte colony stimulating factors. The immediate availability of CRP allowed for preemptive diagnostics and therapy. Real-time knowledge of CRP either changed or significantly influenced therapy in 38% of patients. Study supported in part by ThinkTwice Technologies.
Functional Assessment in Myelodysplasia- Numerical Recovery of Neutrophils May Be Insufficient for Adequate Neutrophil Function.
Authors: Schiller Gary J. , Durna Lara , Koeffler H. P.
Published: October 2019

Myelodysplastic syndromes are a group of oligoclonal hematologic disorders characterized by variable cytopenias and dysplastic changes in the bone marrow. Novel, remittive therapies designed to treat the cytopenias of myelodysplasia, induce apoptosis, differentiation, and antiangiogenesis, in vitro. The success of remittive therapy for myelodysplasia typically depends on clinical criteria of hematopoietic recovery but seldom address functional disturbances. We present a case of an 81 year old man with a one-year history of mild pancytopenia (ANC 0.7 x 103/μL, Hemoglobin 10.3 g/dl MCV 101.7, platelets 51 x 103/μL). A bone marrow biopsy at diagnosis revealed findings consistent with refractory anemia with excess blasts; an initial karyotype revealed no clonal cytogenetic abnormalities. Four months after diagnosis, the patient developed transfusion-dependent thrombocytopenia. A repeat bone marrow biopsy showed equivalent pathology, but cytogenetic analysis demonstrated trisomy 8 in 4 of 20 cells karyotyped. The patient was started on a 12-week trial of paricalcitol, a vitamin D derivative. His treatment course was characterized by persistent moderate pancytopenia and hypercalcemia. Nine months after diagnosis, the patient was admitted to hospital with one week of headache, fever, and visual disturbance. Leukocytes were 28.67 x 103/μL with 11% segmented neutrophils and 70% band forms, Hgb 8.9 g/dl, and platelets 73 x 103/μL. Over the first 24 hours, the patient developed rapidly progressive proptosis leading to MRI of the skull that revealed an abnormal signal in the left frontal lobe. A CT 2 days later revealed extensive sinusitis with a left orbital mass. At resection, rhinocerebral mucormycosis involving the paranasal sinuses, orbit, and cerebral cortex was identified. We performed neutrophil function studies which showed two distinct populations; 59% showed normal oxidative burst capacity, but 41% showed impaired capacity. NBT test also sugested a broadened population of stimulated neutrophils. By light microscopy, numerous pale, agranular, Pelgeroid cells were visible. Despite extensive resection and systemic antibiotics, the patient expired on the 25th hospital day. This case illustrates that normal neutrophil number, in the setting of myelodysplasia, may not denote normal neutrophil function. Functional criteria of response should be considered in clinical studies of remittive therapy for myelodysplasia.
Hepcidin Dysregulation in Patients with Type 1 Gaucher’s Disease.
Authors: Hughes Derralynn A. , Li Anna , Keshav Satish , Mehta Atul
Published: October 2019

Gaucher’s Disease is a glycosphingolipid storage disorder where accumulation of glucosylceramide within reticuloendothelial cells results in diverse systemic manifestations. Patients often present with multifactorial anaemia and elevated ferritin which in the context of chronic inflammation may not truly represent their iron status.

We have investigated the iron status and the role of of hepcidin, a negative regulator of iron absorption, in our cohort of patients with type 1 Gaucher Disease. Analysis of haematological indices and haematinic parameters of 57 patients with type 1 Gaucher disease revealed 31% males and 29% females to be anaemic. Of these haematinic parameters were consistent with iron deficiency or anaemia of chronic disease in 45.5% and 27.3% of patients respectively. Measurement of the soluble transferrin receptor (sTFR), which is elevated in iron deficiency and reduced in iron overload, allowed the iron status of patients to be further classified. Of 17 patients in whom the sTFR was measured 64.7% where found to have normal levels, 11.7% had elevated levels indicative of iron deficiency, and 23.5% reduced levels indicating iron overload. The mean ferritin was 79ug/l and 547ug/l in these groups respectively. Levels of serum prohepcidin, the 84 amino acid precursor of hepcidin, were measured using a competitive elisa (DRG diagnostics). Levels of prohepcidin in those patients with normal sTFR were comparable to levels in control subjects: 387.2 +/− 305ng/ml compared to 357 +/1 120 ng/ml. In those patients with elevated sTFR and iron deficiency the prohepcidin level was appropriately suppressed to 112.5 +/− 17.6 ng/ml. Similarly levels in anaemic Gaucher patients were lower than those in patients with normal haemoglobin. However, in those patients with low sTFR and high ferritin the prohepcidin measuring 225 +/−35.3 ng/ml was not appropriately elevated compared to control subjects or Gaucher patients with normal iron status. We hypothesize that storage of glucosylceramide within lysosomes of macrophages interferes with their iron sensing mechanisms and secretion of regulatory cytokines. Iron overload is not appropriately detected and hepatocyte synthesis of hepcidin not upregulated. Progressive accumulation of iron may therefore occur.
Dynamic Defense System Against HMGB1, a Proinflammatory and Lethal Mediator in Severe Infections and Malignancies.
Authors: Ito Takashi , Abeyama Kazuhiro , Kawahara Ko-ichi , Biswas Kamal K. , Uchimura Tomonori , Yamada Shingo , Hashiguchi Teruto , Maruyama Ikuro
Published: October 2019

High Mobility Group box 1(HMGB1) is an abundant DNA-binding protein that acts as a proinflammatory cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Moreover, an increased HMGB1 in the circulation of septic patients may induce multi-organ failure and lethality. However, very recent observations suggest that the protein also acts as an innate adjuvant, stem cell chemoattractant and growth factor. Thus only systemic and circulatory HMGB1 may induce morbidity and mortality, however, localized HMGB1 may have beneficial effects. Therefore, we serially examined the serum HMGB1 level in patients with various diseases, and also evaluated the significance of the protein.

We demonstrate here how HMGB1 is localized and acts as an immune-adjuvant and a repairing factor in damaged tissue. We first established specific ELISA method to measure HMGB1. An increased level of HMGB1 was detected in the serum from patients with sever sepsis, infections, malignancy and so on. However, serum HMGB1 concentrations were fluctuated during the clinical course, and could not be concluded as a lethal mediator as previously reported. Next we investigated the reason of dynamic fluctuations of the protein in the circulation. Based on our findings, we proposed that this fluctuation of HMGB1 concentrations may be mediated by at least following three fashions; 1) proteolytic degradation by plasmin and thrombin, 2) endothelial thrombomodulin(TM) adsorption, and 3) generation of antibody against the protein. We observed that plasmin efficiently degraded HMGB1 into small fragments. However, interestingly the generated fragments of the protein still possess an ability to produce TNFa in macrophages through an undefined pathway. TM binds the protein on its N-terminus lectin-like domain. Binding of HMGB1 to TM resulted in decrement of TM’s cofactor activity to activate protein C by thrombin. HMGB1 bound to TM was gradually degraded by thrombin. These may be a system to localize HMGB1 only in injury sites where TM is down-regulated or disappeared through endothelial-loss. This may exert endothelial defense system against extracellular HMGB1 in severe tissue injury. Another possibility is that the generated antibody against HMGB1 may neutralize the proinflammatory action of the protein. In this context, we found that some of the antibodies against HMGB1 have the characteristics of P-ANCA(perinuclear anti-neutrophil cytoplasmic antibody). This may alter the phenotype of the underlining diseases. In conclusion, we suggest that HMGB1 is not merely a lethal mediator, but a kind of “testament” mediator of cell necrosis or invasive attacks to dendritic cells.
The Effect of IVIG on Fcγ Receptor-Dependent Monocyte Phagocytosis and FcγR Gene Expression.
Authors: Chan Howard H.W. , Li Wei , Su Brenda B. , Denomme Gregory A.
Published: October 2019

BACKGROUND

The mechanism(s) of intravenous immunoglobulin (IVIG) towards inhibition of monocyte phagocytic activity involves the function and/or the expression of inhibitory FcγRIIb in a murine model. To confirm these findings in human monocytes, we used a human monocyte phagocytic model in vitro to study the effects of IVIG on the phagocytic activity and the expression of FcγR genes.

METHODS

Part A: Monolayer Monocyte Phagocytosis Assay

Normal volunteer’s peripheral blood mononuclear cells (PBMC) were isolated from heparin anticoagulated blood by Ficoll-Hypaque (Pharmacia Biotech) density separation. The PBMCs were washed and the monocytes were purified using a magnetic bead-positive selection method with anti-CD14 antibody (Miltenyl Biotec). 105 monocytes were incubated in a microtiter plate at 37°C for 1 hour before exposed to IVIG 0.5 g/L. Anti-D (WinRho) sensitized Rh positive (R2R2) red cells were added to the monocytes at 0.5 hour and 18 hour post-IVIG treatment. After 1 hour incubation with sensitized RBC, monocytes phagocytic activity is measured by chemiluminescence detection with a LumiCount (Packard). The readings were normalized with maximal chemiluminescence signal achieved by the monocytes without prior exposure to IVIG (positive control). Part B: RT-PCR of FcγRIIa and FcγRIIb

After 18 hours of exposure to two different concentrations of IVIG (0.5 and 5 gm/L), monocytes were collected and total RNA was isolated with TRIzol reagent (Invitrogen). 1 μg of RNA was used to generate first strand cDNA using Superscript II RT kit (Invitrogen). FcγRIIa and IIb were amplified with AmpliTaq Gold DNA polymerase system (Applied Biosystems). The PCR products were evaluated by polyacrylamide gel electrophoriesis.

RESULTS

Part A:

Dose-response curves were generated by plotting normalized chemiluminescence against the concentration of anti-D used to sensitize the red cells. Anti-D sensitized red cells were phagocytosed by monocytes in a dose-dependent manner. There is a time-dependent inhibition of monocyte phagocytosis when monocytes were incubated with IVIG at 0.5 gm/L. (Fig. 1)

Part B:

There is no significant difference in the gene expression of FcRγIIb and FcγRIIa in the adherent monocytes after incubating with either low dose (0.5 gm/L) or high dose (5 gm/L) of IVIG for 18 hours. (Fig 2)

CONCLUSION

Delayed inhibition of phagocytic activity with18-hour exposure to IVIG is not directly mediated via the modulation of FcγRIIb gene expression in human monocytes. Other mechanisms, such as intracellular signalling or receptor coupling, might be involved in the delayed inhibitory effects of IVIG.

Figure Figure Figure Figure
Biological Characteristics of T Cells from CD4 Idiopathic Lymphocytopenia Patients Activated and Expanded Using Xcellerate™ Technology.
Authors: Lawrence Joy , Bonyhadi Mark L. , Cunningham-Rundles Charlotte , Berenson Ronald J. , Long Alice
Published: October 2019

Idiopathic CD4 lymphocytopenia (ICL) is characterized by reduced CD4+ T cell counts in the absence of HIV infection or other defined causes. Patients with ICL generally have CD4+ T cell counts < 100/μL and are at risk for infections. Administration of IL-2 has been reported to reduce the risk of infections in some patients, but CD4+ T cell counts remain low. Currently, there is no available therapy to increase the number of CD4+ T cells in these patients.

We have developed the Xcellerate™ Technology, in which T cells are activated and expanded ex vivo from peripheral blood mononuclear cells (PBMC) using microscopic paramagnetic beads conjugated with anti-CD3 and anti-CD28 monoclonal antibodies (Xcyte™Dynabeads®). In patients with low CD4 counts due to HIV or cancer chemotherapy, administration of T cells activated and expanded using the Xcellerate Technology or similar process leads to significant increases in CD4+ T cell counts to normal levels that are sustained over several months.

In the current study, we tested the ability of the Xcellerate Technology to expand T cells from patients with ICL. We collected data on T cell phenotype, cell expansion, activation marker expression, cytokine secretion, T cell receptor (TCR) repertoire diversity, functional potential and response to restimulation. T cells expanded a median of 902 fold (range 281–1529; n=3 patients), and demonstrated typical induction of surface-activation marker expression, including upregulation of CD25 (IL-2R) and CD154 (CD40L). Phenotypic compositions of the ICL donor PBMC were heterogenous both before and after T cell expansion. Two patients had very high levels of CD4−CD8− T cells, and one of these two patients also had very high levels of γδ+ T cells. Each of these populations was maintained throughout expansion. The other patient displayed high levels of circulating CD3+CD56+ NKT cells that did not proliferate as robustly as other T cells during the expansion process. For two patients, flow cytometric analysis revealed a normal pattern of TCR Vβ surface expression while the remaining tissue had a skewed TCR Vβ distribution pattern. Following initial expansion and subsequent restimulation, T cells rapidly re-expressed surface activation markers and cytokines. In these preliminary studies, T cells from patients with ICL were expanded successfully using the Xcellerate Technology, suggesting that this approach might be used for clinical application in this disease.
A New Class of Chemokine Analogs as Useful Research Tools to Study Chemokine Receptor Function and Promising Therapeutic Agents.
Authors: Huang Ziwei , Kumar Santosh , Choi Won-Tak , Madani Navid , Dong Chang-Zhi , Liu Dongxiang , Wang Jun , An Jing , Sodroski Joseph G.
Published: October 2019

Chemokine receptors play important roles in many physiological processes and are implicated in a wide range of human diseases including acute respiratory distress syndrome, allergic asthma, psoriasis, arthritis, multiple sclerosis, cancer, atherosclerosis and most notably AIDS. To enable the applications of chemokine ligands as probes of receptor biology and pharmacology, and inhibitors of diseases mediated by chemokine receptors, a major problem with the lack of receptor selectivity of these natural chemokines must be overcome. In this study, we have developed a chemical approach combining total protein synthesis and modular modifications to generate a new family of unnatural chemokines termed SMM-chemokines (which refer to synthetically and modularly modified chemokines) with designed receptor selectivity and affinity. A proof of the concept has been provided by applying this strategy to transform a very nonselective chemokine vMIP-II into new analogs with enhanced selectivity and potency for CXCR4 or CCR5, two principal coreceptors for HIV-1 entry. Such novel molecules have been shown subsequently to be valuable probes in gaining insights into receptor binding and signaling mechanisms, and as potent inhibitors to prevent HIV-1 entry and infection. These results strongly support the design concept of these SMM-chemokines and suggest that general applicability of this approach for studying and controlling other chemokine receptors and the diseases they mediate might be anticipated.
Chemical Agonists and High Shear Stress Induce Apoptosis in Human Platelets.
Authors: Leytin Valery , Mykhaylov Sergiy , Allen David J. , Miz Lukasz , Lyubimov Elena V. , Garvey Marie B. , Freedman John J.
Published: October 2019

Apoptosis, or programmed cell death, is appreciated as the main physiologic mechanism that regulates cell life-span and serves for controlled deletion of unwanted cells. Since its discovery in 1972, apoptosis was long attributed exclusively to nucleate cells. It took more than 20 years to recognize apoptosis in enucleated cells cytoplasts and anucleate platelets. During the following years, apoptosis has been demonstrated in platelets treated with natural and artificial agonists, in platelet concentrates aged during storage under standard blood banking conditions, and in animal models of suppressed thrombopoiesis and thrombocytopenia. Other studies documented that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analysed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer (CAP-2000, Brookfield Engineering Labs, Inc., Middleboro, MA), we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arterioles levels (10–44 dynes/cm2) to pathologic high levels (117–388 dynes/cm2) occurring in stenosed coronary, peripheral or cerebral arteries. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIb-alpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation into microparticles, whereas physiologic shear stresses are not effective. Platelets subjected to pathologic shear stresses are characterized by impaired platelet function as shown by the absence of ADP-induced platelet aggregation. Apoptosis changes were also induced by the treatment of platelets with calcium ionophore A23187 (10 μM) and thrombin (1 U/mL). Thus, in the present work, we have demonstrated that platelet apoptosis can be induced by chemical stimuli and by mechanical rheological forces (pathologic high shear stresses). Most of shear-induced activation and apoptosis events occur inside of the platelet, including translocation of CD62 from alpha-granules to the platelet surface, depolarization of mitochondrial inner membrane potential, activation of cytosolic enzyme caspase 3, and translocation of phosphatidylserine from the inner to the outer plasma membrane leaflet. These data suggest that the effects of shear stress on platelet activation and apoptosis are mediated by mechanoreceptor(s) that transmit activation and apoptosis signals to the cell interior. The platelet paradigm of apoptosis induced by chemical agonists and shear stresses suggests that apoptotic cytoplasmic machinery may function without nuclear participation.
Both Platelet ADP Receptors Contribute to Microparticle Formation.
Authors: Kahner Bryan N. , Dorsam Robert T. , Kunapuli Satya P.
Published: October 2019

Microparticles are shed from the platelet membrane upon platelet activation, and they contain negatively charged phospholipids such as phosphatidylserine (PS) on their membrane that bind to coagulation factors and aid in clot formation. While the procoagulant nature of microparticles is clear, the relative contributions of platelet agonists to microparticle formation leading to a thrombus are still unclear. Because secreted ADP from the dense granules has been shown to potentiate granule release, GPIIb/IIIa activation, and platelet thrombin generation, we studied the effect of antagonizing the P2Y1 and P2Y12 receptors on microparticle formation from platelets that are stimulated with convulxin and thrombin. It has been previously shown that a deficiency in the P2Y12 receptor causes an increase in bleeding time, and we found prolonged bleeding times with P2Y1 knockout mice when compared to the wild type mice. Because microparticles are a part of the hemostatic process, we set out to see if there was a contribution of the P2Y receptors on agonist-induced platelet microparticle formation. Flow cytometry was used to measure microparticle formation in human blood in the presence or absence of P2Y1 or P2Y12 receptor antagonists. In addition, we measured agonist-induced microparticle formation in wild type mouse blood and compared the microparticle formation to P2Y1 knockout mouse blood microparticle. Using human blood stimulated with thrombin and convulxin we observed a decrease in microparticle count when administering the P2Y1 antagonist MRS2179 by 17%, and the P2Y12 antagonist AR-C69931MX by 28%. Furthermore, there was a decrease in microparticle formation in P2Y1 knockout mouse blood when compared to the wild type mouse microparticle formation by 13%. These results illustrate that both of the P2Y1 and P2Y12 receptors play an intricate role in microparticle formation in vivo, and may be one more effect underlying the extended bleeding times of patients deficient in the P2Y12 receptor or for the slightly increased bleeding of those taking the P2Y12 receptor antagonist clopidogrel.
Agonist Costimulation Increases Recruitment of Platelet Subpopulations to a Procoagulant Phenotype.
Authors: London Fredda S.
Published: October 2019

Previously we determined that platelets stimulated with PAR 1 agonists, even at levels eliciting maximal responses for support of factor Xa generation, showed only a subpopulation (~10%) that exposed binding sites for factor IXa. Now we show that the same subpopulation of platelets supported binding of both factor Xa and factor IXa. While stimulation of platelets with GPVI agonists collagen or convulxin also caused exposure of factor Xa binding sites on a small subpopulation (~10%) of platelets, simultaneous stimulation with PAR 1 and GPVI agonists resulted in synergistic recruitment of platelets to expose factor Xa binding sites (30–60%). In kinetic studies of platelet-supported thrombin generation, platelets preactivated with thrombin or collagen showed roughly equivalent initial rates of prothrombinase activity (5nM/min, 10nM/min, and 12nM/min for low, medium and high agonist concentrations) that increased with reaction time up to 20 minutes reflecting platelet stimulation by the thrombin generated. Platelets preactivated with both collagen and thrombin, even at high concentrations (collagen 50μg/ml and thrombin 2U/ml), supported additive rates of prothrombinase in contrast to the synergy achieved in exposure of factor Xa binding sites. These data indicate that although single strong agonists recruit only a small subpopulation of activated platelets to undergo procoagulant surface changes, co-stimulation acts synergistically to recruit a larger population. It was previously shown that treatment of platelets with 100μM of the protein kinase C inhibitor RO318220 permitted an increased (three-fold) PAR 1-stimulated platelet support of prothrombinase and an almost 20-fold increase in the size of the population exposing annexin V binding sites. We now show that agonist-stimulated platelets pretreated with 100μM RO318220, whether stimulated with PAR 1 or GPVI agonists separately or combined, recruited ~80% of platelets to bind factor Xa. These data suggest that exposure of a procoagulant phenotype is negatively regulated by a pathway containing a target of high concentrations of RO318220, and that both PAR 1 and GPVI pathways normally converge in relief of that regulation.
Use of All-Transretinoic Acid (ATRA) + Arsenic Trioxide (ATO) To Eliminate or Minimize Use of Chemotherapy (CT) in Untreated Acute Promyelocytic Leukemia (APL).
Authors: Estey Elihu H. , Garcia-Manero Guillermo , Ferrajoli Alexandra , Faderl Stefan , Verstovsek Srdan , Kantarjian Hagop
Published: October 2019

Current therapy cures most patients (pts) with newly diagnosed APL. Therefore, by analogy to childhood acute lymphoblastic leukemia, focus reduction of therapy-associated toxicity appears appropriate. Such reduction implies less reliance on anthracyclines, which can, rarely but not inconsequentially, cause sepsis, myelodysplastic syndromes, and cardiotoxicity. An obstacle to eliminating anthracylines is the belief that ATRA-induced remissions are transient, unless anthracyclines are also given. We examined whether combining ATRA and ATO might allow elimination of CT. ATO (0.15 mg/kg/day) began 10 days after ATRA (45 mg/m2 twice daily). In CR, both drugs were to be continued until 6 months from CR date. CT (typically gemtuzumab ozogamycin, GO, 9 mg/m2 one dose) was only to be given (1) during induction (day 1) if the presenting WBC count was > 10,000/ml, (2) if 2 consecutive polymerase chain reaction (PCR) tests for PML-RARa (sensitivity level 10−4, tests repeated at 2–4 week intervals) were positive 3 months from CR date, or (3) for reversion of a negative PCR test to positive on 2 consecutive occasions as above (molecular relapse). 32 patients (median age 45, 13 with WBC > 10,000 = “high risk”, HR) went on study. The CR rate was 28/32 (88%, 11/13, 85% in HR). Median time to CR was 29 days. Three of the 32 pts died before receiving ATO; APL differentiation syndrome occurred in 5 pts. 25/26 evaluated pts were PCR positive at CR. However, 3 months later, 21/21 were PCR negative, with PCR negativity rates of 17/17, 12/13, 9/10, and 5/5 at, respectively, 6, 9,12, and 12–24 months from CR date. Both molecular relapses subsequently had clinical relapses. No other clinical relapses have occurred, with a median time from CR of 7 months (up to 24 months) in the 26 patients alive in 1st CR. GO was substituted for ATO in 5 patients because of toxicity (arrhythmias in 4, asymptomatic in 3). Among the 29 pts who began ATO, the incidence of arrhythmias was 2/2 in African Americans vs. 2/27 in Caucasians (p = 0.01). Recalling that high-risk pts received CT during induction and excluding pts in whom GO was substituted for ATO because of toxicity, rates of PCR negativity are as follows at 3,6,9,12,and 12–24 months from CR date (rates for high-risk patients in parentheses): 17/17(6/6), 14/14(7/7), 10/11(6/7), 8/9(4/5) and 4/4(3/3). Pending further follow-up, cure of untreated APL without (WBC < 10,000), or with only minimal use (WBC > 10,000) of, CT appears feasible.
Comparison of Clinical Outcomes in Patients with Heparin-Induced Thrombocytopenia Treated with Direct Thrombin Inhibitors.
Authors: Francis John L. , Sandhu Ranjeev , Drexler Alane , Dougherty John
Published: October 2019

Heparin-induced thrombocytopenia (HIT) is a clinical syndrome that has been reported to occur in 3–5% of patients treated with unfractionated heparin. If untreated, 36–50% of patients diagnosed with HIT develop life or limb-threatening thromboses. The occurrence of thrombosis, the most common serious complication of HIT, results in rates of amputation and death of 10–20% and 20–30% respectively. Thus, the desired clinical outcomes in patients with HIT are the prevention of thromboembolic complications, limb amputation, and death. The three direct thrombin inhibitors available in the U.S. are lepirudin, argatroban and bivalirudin. Lepirudin and argatroban have both been shown in clinical trials to significantly decrease the incidence of these complications in patients with HIT. Our institution also has experience with bivalirudin as treatment for HIT. We therefore sought to confirm whether all-cause mortality, length of stay, bleeding rate, time to platelet recovery, absolute change in platelet count following therapy, and percentage of therapeutic APTTs differed among patients treated with these agents at our institution. Data were collected by retrospective chart reviews, and from the Florida Hospital pharmacy computer system. Length of stay was calculated as the time to hospital discharge following the finding of a positive heparin-platelet factor 4 antibody test. For the purposes of comparison, the therapeutic range for APTT was taken as 50–90 seconds. As shown in the table, there were no statistically significant differences in any of the endpoints, when the efficacy and safety of the three direct thrombin inhibitors were compared. It therefore appears that within our institution, each of the direct thrombin inhibitors are equally efficacious and safe in treating the clinical syndrome of HIT, with similar outcomes with respect to length of stay, recovery of platelet counts, incidence of bleeding, and overall mortality. We conclude that selection of a direct thrombin inhibitor can be guided by the patient’s clinical status and organ function instead of efficacy and safety considerations.

Drug n All-cause mortality (%) Length of stay (days) Bleeding (%) Time to platelet recovery (days) Therapeutic APTT (%) Lepirudin 7 28.5 11.0 28.5 5.3 70 Argatroban 20 30.0 15.1 20.0 5.2 65 Bivalirudin 24 25.0 18.7 25.0 5.4 74 P-value NS NS NS NS NS
Treatment of New Cases of Acute Promyelocytic Leukaemia by Arsenic Trioxide.
Authors: Ghavamzadeh Ardeshir , Alimoghaddam Kamran , Ghaffari Hamidolah , Rostami Shahrano , Mortazavi Yousef , Mousavi Asadolah , Tootonchi Mehrangiz , Jahani Mohamad , Hosseini Roholah , Iravani Masoud , Bahar Babak , Aghdami Nasser
Published: October 2019

Purpose: Arsenic Trioxide approved for treatment of relapsed or refractory APL to ATRA. We studied the effects of Arsenic Trioxide as first line treatment of new cases of APL and their follow up.

Material and methods: we studied 73 new cases of APL diagnosed by morphologic criteria and confirmed by cytogenetic, RT-PCR for PML/RARA and/or FISH and followed patients for MRD by sensitive nested RT-PCR.

Our patients were 30 males and 43 females with median age 30+/− 12. Patients treated by infusion of 0.15mg/kg/d of Arsenic Trioxide to complete remission by morphologic criteria or till day +60. In patients who complete remission achieved, after 28 days rest, again we began Arsenic Trioxide 0.15mg/kg/d for 28 days as consolidation.

Results: complete remission were achieved in 66 patients( 90.4%) and 7 early mortality. Median time to complete remission was 30+/−6.4 days. Most common cause of mortality was APL maturation syndrome ( 4 cases)

Most common toxicities during induction phase were, APL maturation syndrome (10 cases), serositis(6 cases) and hepatotoxicity (19 cases).

63 cases(86.3%) are alive with a median follow up of 17+/−12.65 months. 14 relapses observed in our patients and complete remission achieved with re-treatment by Arsenic trioxide in 11 of them. Also we could control 3 fatal bleeding by infusion of activated factor 7(NovosevenÒ) which stopped hemorrhage .

One year and two/three years survival of patients were 86% and 84%. Most common cause of death was APL maturation syndrome in 4 patients and relapse in 3 cases.Conclusion: Arsenic Trioxide is acceptable as first line treatment of APL and its result is comparable to ATRA with chemotherapy.
Clinical Associations and Significance of Biphasic aPTT Transmittance Waveforms.
Authors: Stoffman Jayson M. , Israels Sara J.
Published: October 2019

Many modern automated coagulometers determine coagulation parameters such as the PT and aPTT using optical endpoints. One instrument (MDA-180, BioMerieux, Durham, NC) continuously monitors changes in optical transmittance through the specimen over the course of clot formation, and is able to analyze measurements derived from the resulting waveform. The waveform is typically sigmoid, with a sharp drop in transmittance at the time of clot formation. In some specimens, a biphasic waveform (BPW) pattern has been observed, in which the normally flat pre-coagulation segment has a negative slope prior to the sharp drop in transmittance associated with clot formation. The BPW pattern has been associated with sepsis, DIC, and increased mortality in adult patients.

The Special Haemostasis Laboratory of the Health Sciences Centre in Winnipeg, Canada uses the MDA-180 for all automated coagulation assays. Over a two-year period (August, 2002 to July, 2004), a BPW was identified in 21 adult and 12 pediatric patients. Hospital records of these patients were reviewed for details of their clinical condition, and coagulation test results were examined for presence of hemostatic abnormalities.

Sepsis was a common diagnosis at the time of detection of the BPW, seen in 57% of the adults and 42% of the children. However, 4 adults (19%) and 4 children (33%) were identified during routine investigations for clinical bleeding or thrombophilia. In contrast to previously reported studies, DIC was less prevalent, seen in only 7 (33%) of the adults and 3 (25%) of the children. The presence of lupus anticoagulants, alone or in conjunction with other coagulation abnormalities, was observed in 8 adults (38%) and 9 children (75%). This association has not previously been reported. Nine adults (43%) and four children (33%) died, all from complications of sepsis.

Despite the small sample size, this study questions the clinical utility and specificity of the BPW as an indicator of sepsis and DIC. The association with DIC seems particularly weak in the pediatric population, in which the BPW has not been previously evaluated. Further study is required to determine the strength of the association between the BPW and DIC, and to identify other clinical and laboratory conditions, such as lupus anticoagulants, which may confound this association.
Factor V Leiden Mutation Enhances Thrombin Formation in Healthy Newborn Infants.
Authors: Hyytiäinen Satu , Ulander Veli-Matti , Wartiovaara-Kautto Ulla , Kaaja Risto , Heikinheimo Markku , Petäjä Jari
Published: October 2019

Background: At birth, the coagulation system is activated resulting in thrombin formation in the newborn. In sick newborns, the coagulopathy may lead to bleeding or thrombotic complications. Known genetic prothrombotic defects are risk factors for thrombosis in adults but their potential influence on perinatal coagulopathy remains unknown. Accordingly, we prospectively studied the influence of genetic thrombophilia on thrombin formation during the first two weeks of life.

Methods: Newborns from 22 pregnant women with thrombophilia (Factor V Leiden (FVL), n=16; anticardiolipin antibodies, n= 3; lupus anticoagulant, n=1; protein C deficiency, n=1; prothrombin G20210A mutation, n=1) were enrolled into the study. All women were treated with low-molecular-weight heparin during pregnancy and all the infants were born at term. Blood samples were collected from the umbilical cord, and on postnatal days 1 and 14. Newborns were screened for FVL and prothrombin G20210A mutation. Eight FVL heterozygotes were found. No infant tested positive for anticardiolipin antibodies or lupus anticoagulant. Thrombin formation was assessed by measuring prothrombin fragment F1+2. For samples on day 1, a previous cohort of 32 newborns (mean gestational age 33.6 weeks, unknown FVL status) treated in our neonatal intensive care unit (NICU) was used as an additional control.

Results: Relative to known normal range in adults, enhanced thrombin formation (F1+2>1.1 nM) was detected in cord blood and on day 1 in all newborns followed by near normalization on day 14. The median level of F1+2 from umbilical cord sample to day 1, and to day 14 in FVL negative newborns was 2.4 nM (n=13) − 2.0 nM (n=10) − 2.0 nM (n=12), and in FVL heterozygotes 4.8 nM (n=7) − 5.8 nM (n=5) − 1.7 nM (n=8). In FVL heterozygotes but not in FVL negative newborns the F1+2 decrease from umbilical cord sample to day 14 was significant (Wilcoxon p=0.02). On day 1, the FVL heterozygotes showed elevated F1+2 levels when compared with a cohort of 32 newborns in NICU (median F1+2 2.3 nM in NICU group vs 5.8 nM in FVL heterozygotes; p=0.06).

Conclusions: Factor V heterozygosity was associated with transiently enhanced thrombin formation in healthy newborns during the first days of life.
Murine Reagents and Analytical Tools to Enhance the Utility of Mouse Models for the Study of Human Thrombotic Disorders and Disease States.
Authors: Marcotuillo Michelle , Johnson Vicki , Jenny Richard J. , Dorfman Ryan H.
Published: October 2019

The mechanisms that govern blood coagulation and fibrinolysis have been studied extensively by employing a variety of experimental techniques and in-vivo model systems. Murine models are a valuable resource to test new strategies in the areas of thrombosis and haemostasis. Because both normal and genetically altered mice are available, the murine models are ideal for this application. Murine models are in prevalent use and there exists a limited number of reagents available to assist the researcher. Development of murine reagents and analytical tools will enhance the utility of murine coagulation models by allowing researchers to acquire appropriate standards for characterization, validation, and screening of murine model systems. Recently, we have purified and characterized a group of nine mouse plasma proteins including the enzymatic forms of many of these proteins. A total of eight new polyclonal antibodies to murine antigens were generated including sheep a-mouse plasminogen and sheep anti-mouse factor X. Here we report on the development of two competitive based ELISA’s for the quantitation of mouse plasminogen and mouse factor X. The assay format utilizes purified antigen (plasminogen and factor X) standard. A standard curve is generated based upon the competition between fluid-phase antigen and an antigen-biotin conjugate for an immobilized affinity purified polyclonal antibody (sheep a-mouse plasminogen and sheep anti-mouse factor X). Solid-phase antibody/biotinyl-antigen complexes are detected using avidin-peroxidase. A colormetric signal, which is inversely proportional to the amount of antigen in the fluid-phase, is obtained following the addition of chromogenic substrate. Calibration curves are generated for an immobilized antibody ELISA by plotting the absorbance at 490 nm versus the concentration of non-biotin labeled antigen standard. Biotinylated-antigens were used at single concentrations of 50 ng/ml and 10 ng/ml for factor X and plasminogen respectively. In both cases, immobilized antibodies were adsorbed to microtiter plates at 5ug/ ml. Linearity of the factor X assay extends from approximately 10 ng/ml to 1000 ng/ml. The intra-assay coefficient of variation (CV) is ≤1.1 %, while the inter-assay CV is ≤5 %. Linearity of the plasminogen assay extends from approximately 0.1 μg/ml to 10 μg/ml. The intra-assay coefficient of variation (CV) is ≤2.0 %, while the inter-assay CV is ≤6.3 %. Preliminary studies have demonstrated the feasibility of these assays. With further testing and analysis these assays may prove useful to the researcher attempting to quantitate mouse plasma concentrations of these analytes.