bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment with Rituximab, CHOP and Highly Active Antiretroviral Therapy (HAART) in AIDS-Related Diffuse Large B-Cell Lymphomas (DLBCL). Study of 60 Patients.
Authors: Ribera Josep-María , Oriol Albert , Morgades Mireia , González-Barca Eva , Miralles Pilar , Lopez-Guillermo Armando , Lopez Andres , Xicoy Blanca , Abella Eugenia , Gardella Santiago , García Marta , Bargay Joan , Perez-Equiza Katy , Briones Javier , Rodriguez Lluis , Provencio Mariano , Escoda Lourdes , Soler Alfons , Canales Miguel , Asensio Antonio , Romero Antonio , Ferrer Secundino , Feliu Evarist
Published: October 2019

Background and objective. Rituximab and CHOP (R-CHOP) is the most employed treatment for DLBCL, but pts with AIDS-related lymphomas are usually excluded from clinical trials. The objective of this open, prospective multicenter trial is to evaluate the feasibility, efficacy and toxicity R-CHOP and HAART in AIDS-related DLBCL.

Patients and methods. Between April 2001 and July 2005, 60 consecutive HIV-infected pts with newly diagnosed DLBCL were included in 20 Spanish hospitals. HAART was given to all patients from diagnosis if they were not already receiving it. Six cycles of R-CHOP were administered, IT CNS prophylaxis (MTX, ARA-C and hydrocortisone) was given in every cycle to all patients. G-CSF support was recommended Response to chemotherapy, toxicity, OS and DFS for complete responders were recorded.

Results. Median age 42 yr (range 26–64), 49 (82%) males, 30 (50%) with previous known diagnosis of HIV infection (median from dx HIV to NHL 10 yr, range 0.5–19). Median CD4 lymphocyte count 152/mL (range 0–905), median HIV load 19x103 copies/mL (range 0–2x106). 36 pts were receiving HAART at the time of NHL dx (median 3.5 yr, range 0.5–9). Extranodal involvement 43 (72%), stage III-IV 38 (63%) and 36/56 had intermediate-high or high age-adjusted IPI score. 10 patients are under treatment, 1 (2%) withdrawal, 6 (12%) induction death (infection 3, hepatic failure 2, multiorgan failure 1), 10 (20%) resistant, CR 33 (66%). After a median follow-up of 2 yr, 2-yr survival probability was 63% (95%CI 50–76). The probability of remaining alive and in first CR at 2 yr for complete responders was 89% (95%CI 77–100). Three patients died in first CR (opportunistic infection, sudden death and violent death) and no relapses have occurred to date. Virologic and immunologic responses to HAART at 6 months after the completion of treatment were maintained or achieved in 17/21 (81%) and 14/22 (64%) of patients, respectively. Out of 245 R-CHOP cycles analysed the most frequent grade II-IV toxicities were infections (30, 12%), gastrointestinal (21, 9%) and neurologic (5, 2%).

Conclusion. In patients with AIDS-related DLBCL the combination of HAART and R-CHOP is feasible and effective. In this trial the response rate and survival are comparable to those obtained in immunocompetent patients treated with R-CHOP.
Time to Treatment Failure (TTF) and FLIPI Correlate Well with Survival and Are Independent Variables in Stage IV Indolent Follicular Lymphomas (IFL).
Authors: Fayad Luis , Pro Barbara , Liu Qi , Hess Mark , Hagemeister Fredrick , Romaguera Jorge , Samaniego Felipe , Cabanillas Fernando , Rodriguez Alma , Younes Anas , Wang Michael , McLaughlin Peter
Published: October 2019

BACKGROUND: The FLIPI score has been shown to predict survival in FL pts. Previous studies have suggested that type of treatment, and quality of response may not affect survival in pts with stage IV IFL. We address here the significance of TTF and quality of response as prognostic factors for survival in stage IV IFL.

METHODS: We reviewed the records of 580 pts with stage IV IFL treated from 1972- to 2002 with anthracycline containing regimens +/− rituximab. Demographics: male 51%, age 60 28%, Hgb<12 17%, high LDH 23%, 5 or more nodal groups 66%, BM+ 93%, FLIPI 3 or more 76%.

RESULTS: With a median f/up of 7 years

Response <PR (n=17) PR (n=86) CR (n=477) % dead 88 74 34 Median OS (mos) 19* 56* 185* TTF <1yr (n=60) 1–2yr (n=88) >2yr (n=432) % dead 85 53 33 Median OS (mos) 25* 76* 191* *p=0.001 % ORR (% CR) TTF (mos) Median OS (yrs) FLIPI<3 (n=151) 98 (89) 84 20.5 FLIPI≥3 (n=429) 96 (80) 44 10

Regression analysis for survival showed that both TTF and FLIPI correlated well with survival and were independent of each other (p<0.00001).

CONCLUSION: As expected, FLIPI <3 correlated with longer survival in IFL treated with anthracycline containing regimens. Interestingly, achievement of CR, longer response duration as measured by TTF also correlated with longer survival.
Meta-Analysis of Impact of Epoetin Alfa Versus Darbepoetin Alfa on Blood Transfusion Requirements.
Authors: Lefebvre Patrick , Moyneur Erick , Zilberberg Marya , Duh Mei S. , Aggarwal Jyoti , Piech Catherine Tak
Published: October 2019

Background: Epoetin alfa (EPO) and darbepoetin alfa (DARB) are commonly used for the treatment of chemotherapy-induced anemia (CIA). To date, three randomized head-to-head studies have compared EPO dosed 40,000 Units once weekly (QW) and DARB dosed 200mcg once every two weeks (Q2W). As transfusion results across these studies were inconsistent a meta-analysis was undertaken to provide a pooled estimate of the comparative difference in red blood cell transfusion rates between DARB and EPO treatment groups.

Methods: Demographic and transfusion rate data were extracted from the three publicly available head-to-head comparison studies (pooled N=940 for DARB and N=933 for EPO). Odds ratios, risk ratios and risk differences of transfusion rates from week 1 to the end of treatment between DARB and EPO groups were analyzed using both fixed- and random-effects meta-analysis methods; the latter incorporated between-study variation in addition to within-study variation. A multivariate logistic regression model weighted by the number of patients in each study arm and including population-level characteristics from each study arm, was conducted to isolate the treatment effect on the risk of transfusion after controlling for differences in baseline characteristics between DARB and EPO patients.

Results: Fixed- and random-effects models generated similar results (p>0.42 for homogeneity test across studies), both demonstrating that the DARB Q2W group had a statistically significantly higher transfusion rate than the EPO QW group. Using the random-effects model, the odds ratio of transfusion in the DARB group was 1.28 of that in the EPO group (95%CI: 1.03– 1.59). The corresponding risk ratio and risk difference were 1.21 (95%CI: 1.02–1.43) and 4% (95%CI: 0.3%–8%), respectively. Multivariate regression analysis further confirmed DARB Q2W had a higher transfusion rate compared to EPO QW, after controlling for age, baseline hemoglobin and the proportion of breast cancer patients (adjusted odds ratio=1.45, 95%CI: 1.10–1.91).

Conclusion: This meta-analysis demonstrates that, in patients with CIA, EPO 40,000 units QW is more effective at reducing RBC transfusion requirements than DARB 200 mcg Q2W.
Investigation of Underlying Reasons of Factor VIII Deficiency in Haemophilia A Patients with Undetectable Mutations in the Factor VIII Gene.
Authors: El-Maarri Osman , Schröder Jörg , Klein Claudia , Schwaab Rainer , Goodeve Anne , Negrier Claude , Thompson Arthur , Srivastava Alok , Oldenburg Johannes
Published: October 2019

Haemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the coagulation factor VIII (F8) gene. Despite applying sensitive methods for mutation detection, and after excluding the inversions mutations a causative mutation is not identified in F8 gene in about 2,5% of severe HA patients (53 patients out of 2350 German patients). Analysis of mRNA from a small group of such (German) patients has excluded mutations deep in the introns that may affect normal splicing or mechanisms causing some unknown rearrangements of the F8 gene as the cause of HA. Among this group, in one patient no F8 mRNA was detected. Using two common polymorphisms in F8 exon 14, we were able to show that the same allele shared by the patient, his mother and his sister was not detected by reverse transcription PCR (RT-PCR) from total blood mRNA. These findings strongly suggest that the cause of HA in this patient is either absence or rapid degradation of the F8 mRNA which points to a novel mechanism leading to HA. To further investigate the cause of Hemophilia A in these patients an international multi-center study was established. The aim of this study to assemble a large collection of such families that could provide clues for novel factors/mechanisims that are important for the F8 molecule biogenesis and protein regulation as well as F8 expression. This multi-center study is therefore suposed to identify novel mechanisms causing HA.
The Bcl-2 Family Inhibitor ABT-263 Shows Significant but Reversible Thrombocytopenia in Mice.
Authors: Shoemaker Alex R. , Mitten Michael J. , Adickes Jessica , Oleksijew Anatol , Zhang Haichao , Bauch Joy , Marsh Kennan , Frost David J. , Madar David , Tse Christin , Fesik Stephen W. , Rosenberg Saul H. , Elmore Steven W.
Published: October 2019

ABT-263 is an orally bioavailable small molecule inhibitor of Bcl-2 family proteins with a Ki of ≤ 1 nM against Bcl-2, Bcl-XL, and Bcl-w. Potent cytotoxic effects have been demonstrated against small cell lung carcinomas and lymphomas (EC50 of less than 500 nM in vitro against numerous cell lines). Tumor growth stasis and regression in xenograft models confirmed this activity. However, a unique thrombocytopenia has been observed in mice, rats, and dogs. Using a mouse model to characterize the extent and duration of decreased circulating platelets, outbred CF-1 or inbred scid-bg mice were treated with increasing doses of ABT-263. Blood samples were collected at various times post dosing and platelet numbers were determined using automated hematology analysis. The results showed a rapid, dose-dependent reduction in circulating platelet counts with a platelet nadir (>80% reduction relative to baseline) occurring approximately 6 hours after a single 100 mg/kg oral dose of ABT-263. Platelet counts returned to normal levels by 72 hrs post-dose. Analysis of bleeding times using a tail-nick method demonstrated a correlation between reduced platelet count and increased bleeding time. At doses of 50 mg/kg and higher, bleed times were increased by more than 5-fold relative to vehicle controls. In multi-dose studies, platelet levels did not continue to decline relative to the first dose of ABT-263. Mice treated with ABT-263 at 100 mg/kg/day for as long as 21 days exhibited no overt signs of toxicity (bleeding, significant weight loss, etc). Because the spleen is a potential site for clearing of damaged or aggregated platelets, a study using splenectomized mice was also conducted. The kinetics of platelet reduction and recovery were similar in splenectomized vs. intact mice with no additional adverse events noted. Evaluations of plasma drug levels associated with preclinical anti-tumor efficacy vs. thrombocytopenic effects indicate that a reasonable therapeutic window exists for ABT-263.
Evidence That the MPL 515L and 515K Mutations Occur in a Lympho-Myeloid Progenitor in Idiopathic Myelofibrosis and Are Associated with TPO Hypersensitivity.
Authors: Chaligne Ronan , Le Couedic Jean-Pierre , Tonetti Carole , Besancenot Rodolphe , Wagner-Ballon Orianne , Debili Najet , Vainchenker William , Giraudier Stephane
Published: October 2019

The MPL 515L mutation has recently been described in 10% of Idiopathic Myelofibrosis (MMM) as an essential oncogenic event. Here we report another MPL mutation in the same codon of MPL (MPL515K). The MPL515L mutation has been detected in granulocytes but has not been yet detected in patient hematopietic stem cells. This raises the question whether this molecular event occurs in a true stem cell (ie: lymphoid/myeloid progenitor cell), as it has already been shown for JAK2 V617F occurrence in MMM and PV.

The aim of this work was to study the presence of the mutation and another MPL mutation (MPL 515K) in both myeloid and lymphoid lineages in JAK2 V617F negative MMM. We therefore looked for the mutation first in mature myeloid and lymphoid cells and second in lymphoid/myeloid progenitors progeny after CD34+ and CD34+CD38- cell isolation from peripheral blood. Three IMF, patients harbouring MPL mutations were enrolled in this study after informed consent. Peripheral blood granulocytes and platelets were purified by standard methods and B, T, NK cells and monocytes were solated by combined immunomagnetic and flow cytometric procedures. The same techniques were used to sort CD34+ and CD34+CD38- subpopulations from peripheral blood. Clonal B/NK/Myeloid differentiation from CD34+CD38- cells and T cell differentiation from CD34+ cells were performed respectively onto a MS5 layer in the presence of SCF, FLT3L, IL2, IL3, IL7, IL15, TPO and in Fetal Thymic Organ Cultures (FTOC). Genotyping of mature cell populations, B/NK/Myeloid clones and CD34+ derived T cells were performed by direct sequencing. Moreover, CD34+ cells were cultured in a one cell per well experiment to determine the sensitivilty of these patient’s cells to low dose TPO.

The MPL mutations were present in granulocytes and platelets from all patients, and in monocytes. We detected the mutation in B and NK cells in all three cases. The MPL 515L and MPL 515K mutations could be subsequently detected in CD34+ cells and in B/NK/Myeloid and/or NK/myeloid CD34+CD38- derived clones from all IMF patients. Interestingly, MPL 515L homozygous clones were detected in B/NK/Myeloid and/or NK/Myeloid clones from 1 MMM patient. Using the FTOC assay, the mutation was also detected in all T cell fractions derived from CD34+ cells from 2 MMM patients. At least we found that MPL mutations induce a spontaneous megakaryocytic growth in cell culture but also a hypersensitivity to low dose TPO.

These results demonstrate that the mutation occur in a lymphoid/myeloid progenitor cell in MPL 515L or K MMM and give rise to a hypersensitivity to TPO. Thus, these MPD take their origin in a true lymphoid/myeloid progenitor cell. In accordance with mouse model, this represent a good argument for a causative role of MPL mutations in MMM.
Effect of Valproate on In Vitro Steroid Responsiveness in 2-Phase Erythroid Culture in Diamond Blackfan Anemia (DBA): Potential Role for Histone Deacetylation in the Mechanism of Action of Steroids in DBA.
Authors: Ohene-Abuakwa Yaw , Marius Carine , White David , Sarah Ball
Published: October 2019

We have previously adapted a 2-phase liquid erythroid culture system to demonstrate a consistent and profound failure in erythroid expansion in DBA cultures, which localizes to early in the second erythropoietin-dependent phase of culture. We have shown a partial corrective effect of steroids, which both enhance the clonogenicity of early erythroid colony forming cells, and increase erythropoietin sensitivity by a direct effect on erythroid progenitors at the later stage of CFU-E expansion. Following a report of the induction of remission by valproic acid in a patient with refractory DBA, we have extended our study to include the effect of added valproate at concentrations equivalent to therapeutic plasma levels. In preliminary studies of cultures from normal controls, sodium valproate at 50ng/ml markedly increased erythroid expansion in cultures without added dexamethasone (p<0.001, n=13). A similar effect was observed in DBA cultures, although this was not statistically significant (p=0.07, n=7). However, the enhancing effect of 10−7M dexamethasone in control cultures was inhibited by valproate 100 ng/ml, (n=13, p<0.05). A similar inhibition of the steroid effect was observed in both of 2 DBA cultures showing in vitro steroid responsiveness. In contrast, at low dose valproate (25ng/ml) and dexamethasone (10−8M), there was evidence of synergistic enhancement of erythroid expansion. Valproate is a known histone deacetylase (HDAC) inhibitor, and its interactions with dexamethasone in this culture system strongly suggest that dexamethasone influences erythroid histone acetylation status. In support of this, a similar dose-dependent interaction with dexamethasone was observed with trichostatin A, another HDAC inhibitor. This represents a potential molecular basis for the mechanism of action of the stimulatory effect of steroids in erythropoiesis, although the complex dose-dependent interaction between valproate and dexamethasone would be consistent with differential effects of steroids on histone deacetylation at different stages in erythropoiesis. Interestingly, the inhibition of the effect of dexamethasone at higher concentrations of valproate resembles our previously reported effect of prolactin in this culture system, suggesting that modification of histone acetylation also underlies the effect of prolactin. We will address this further by analysing the histone acetylation status of primary erythroid cells throughout the 2-phase culture system, especially at the time of divergence of DBA and control cultures, and the effect of different concentrations of steroids. This may elucidate the individual variation in steroid responsiveness in DBA, and possibly suggest steroid sparing agents for clinical use.
pak2a Mutations Cause Cerebral Hemorrhage in Redhead Zebrafish.
Authors: Buchner David A. , Shavit Jordan A. , Su Fengyun , Yamaoka Jennifer S. , Kamei Makoto , Mcgee Beth , Hanosh Andrew W. , Weinstein Brant M. , Ginsburg David , Lyons Susan E.
Published: October 2019

Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog of the p21-activated kinase Pak2. This IVS9 T+2A mutation renders greater than 90% of transcripts nonfunctional, resulting in a hypomorphic allele. Surveying zebrafish expressed sequence tag databases, we identified two Pak2 orthologs in zebrafish, designated Pak2a and Pak2b, with the redhead mutation in pak2a. Central nervous system (CNS) hemorrhage in redhead embryos was rescued by injection of wild type pak2a mRNA. Morpholino knockdown of pak2a in wild type fish phenocopies the redhead mutant, but with an increase in penetrance and severity, including hydrocephalus and pericardial edema secondary to severe hemorrhage. Injection of either pak2a or pak2b mRNA was able to rescue this phenotype. In addition, pak2b knockdown worsened the bleeding phenotype in redhead embryos, with no effect on wild type fish. These results suggest a partial overlap in function between Pak2a and Pak2b. Confocal microscopy was performed at 2.5 dpf in pak2a knockdown embryos transgenic for Gata1-dsRED and Flk1-eGFP, distinctively labeling erythrocytes and endothelial cells, respectively. The sites and magnitude of hemorrhage were variable between individual embryos, but there were no obvious abnormalities in vessel patterning. In summary, we have identified a critical role for Pak2 in maintaining CNS vessel integrity in zebrafish, without apparent effects on other vascular beds. Further analysis of this signaling pathway in the vessel wall may provide novel insight into the mechanisms underlying vascular heterogeneity in mammals, and the tissue specific function of Pak2 in the CNS vasculature.
Notch1 Provides a Proliferative Signal in Mouse T Cell Leukemia by Directly Targeting c-Myc.
Authors: Draheim Kyle M. , Sharma Vishva M. , Calvo Jennifer A. , Cunningham Leslie A. , Hermance Nicole , Krishnamoorthy Veena , Beverly Levi , Bhasin Manoj , Capobianco Anthony , Kelliher Michelle A.
Published: October 2019

As demonstrated in mouse models and in primary human T cell leukemic samples, gain of function mutation(s) in Notch1 is a common genetic event in T cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus where it forms part of a transcriptional activator complex. We have demonstrated that mouse leukemic growth is Notch1-dependent, since treatment with γ-secretase inhibitors (GSI) results in rapid cell cycle arrest and/or apoptosis. To specifically identify Notch1 target gene(s) in leukemia, we developed mouse T cell leukemic cell lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel and direct Notch1 target gene. Consistent with these findings, retroviral insertional mutagenesis screening of our tal1 leukemic mouse model reveal common insertions in either notch1 or c-myc. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with GSI treatment or Notch1 inhibition in 83% mouse tal1 leukemic cell lines tested. Yet in a subset of leukemic cell lines, retroviral expression of c-myc fails to rescue leukemic growth, whereas expression of intracellular Notch1 in these lines remains capable of restoring growth. These data suggest that additional Notch1 target genes other than c-myc contribute to leukemogenesis. Other Notch1 target genes in thymocyte developement and their potential role in leukemogenesis will be discussed.
Platelet Microparticles, Platelet Activation and Platelet-Leukocyte Conjugates in HIV Infected Patients with Optimal Response to Highly Active Antiretroviral Therapy (HAART).
Authors: Corrales-Medina Vicente F. , Simkins Jacques , Chirinos Julio A. , Lopez-Sanabria Bernardo , Symes Stephen , Jayaweera Dushyantha T. , Jy Wenche , Jimenez Joaquin J. , Horstman Lawrence L. , Ahn Yeon-Soong
Published: October 2019

Background: HIV infection and its treatment have been associated with an increased risk of atherothrombotic cardiovascular disease. Little is known about the status of platelets in optimally controlled HIV infection. We aimed to assess the status of platelets using flow cytometry markers of platelet activation/apoptosis in HIV-infected patients who demonstrated an optimal response to highly active antiretroviral therapy (HAART).

Methods: In the current study we measured P-selectin expression in platelets, platelet microparticles (PMP) and platelet-leukocyte conjugates (PLC) by flow cytometry in 29 patients (mean age=42.1±7 years) with HIV infection on HAART who demonstrated an optimal viral and immunological response (CD4+ cell count > 200 /mm3 and undetectable viral load by ultrasensitive PCR analysis). Patients with a history of diabetes, smoking, illicit drug use, thrombotic, cardiovascular or malignant disease were excluded. We used age- and gender-matched healthy individuals as controls. PMP were measured by flow cytometry based in the co-expression of CD31 and CD42b. Platelet activation was also assessed by expression of activation marker P-selectin (expressed in fluorescence intensity units, FIU). PLC were measured based on the co-expression of CD45 and CD41 (expressed as percentage of leukocytes positive for CD41).

Results: Platelet expression of P-selectin was not significantly different between HIV infected patients (7.35 FIU; IQR=4.00–15.43) and controls (13.32 FIU; IQR=4.91–23.02; p=0.11). However, the levels of PMP were markedly elevated in HIV-infected patients (24,951 (counts/μl; IQR=17,887–36,175) compared to the control group (13,862 counts/μl; IQR=9,984–20,736; p=0.001). The levels of PLC were lower in HIV-infected patients (52.2%; IQR=42.65–65.75) compared to controls (63.8%; IQR=56.9–78.7; p=0.0007). There was a trend for a negative correlation between PMP levels and the CD4 count (Pearson R=−0.34; p=0.07).

Conclusions: HIV infected patients with optimal response to HAART demonstrate an increased number of circulating free PMP, normal levels of platelet expression of activation marker P-selectin and decreased levels of PLC. A decreased number of PLC indicates decreased binding of platelets and PMP to leukocytes in HIV-infected patients, which could be partially responsible for increased levels of free PMP. In addition, PMP (unlike P-selectin expression) may be a sensitive marker indicating low-grade platelet activation in HIV infection. Increased PMP levels are likely to contribute to the atherothrombotic risk HIV+ infected patients on HAART.
Utility of Optical Density Values from Heparin-Platelet Factor 4 Antibody Testing and Probability Scoring Models To Diagnose Patients with Heparin Induced Thrombocytopenia.
Authors: Janatpour Kim A. , Gosselin Robert C. , Dager William E. , Lee Andrew , Owings John T. , Wun Ted
Published: October 2019

Background: Heparin induced thrombocytopenia (HIT) is a potentially life threatening complication of heparin administration caused by antibodies directed to the heparin-platelet factor 4 (PF4) complex and characterized by thrombocytopenia with a seemingly paradoxical high risk of thrombosis. Diagnosis is challenging, and is based on both clinical suspicion and laboratory detection of heparin-PF4 antibodies. The Warkentin 4 T’s “pre-test” probability and Chong’s “post-test” probability models have been developed to aid the diagnosis of HIT. Enzyme-linked immunoabosorbant assay (ELISA) laboratory measurement of heparin-PF4 antibodies is commonly used but has a low positive predictive value for thrombosis. Recent reports suggest that using an ELISA optical density (OD) value of ≥ 1 may improve the predictive value for thrombosis.

Methods: We performed a retrospective analysis of 105 patients with suspected HIT who were treated with a direct thrombin inhibitor and evaluated the anti-heparin-PF4 ELISA OD values and Warkentin 4 T’s scores for sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) using the Chong’s score to define HITBoth the manufacturer’s OD threshold of 0.4 and ≥ 1 were evaluated.

Table 1. Comparison of sensitivity, specificity, and predictive values for 4 T’s and OD levels, alone and in combination Sensitivity (%) Specificity (%) NPV (%) PPV (%) For calculations, Chong’s categories of definite/probable and unlikely were used to define presence or absence of HIT. 4T’s score (high and low) 81 100 80 100 OD level 0.4 69 75 65 78 OD level ≥1 38 85 52 77 4T’s + OD 0.4 94 100 94 100 4T’s +OD ≥1 69 85 81 75

Results:

The sensitivity, specificity and predictive value of ELISA alone were inferior to the 4 T’s clinical scorings system. The sensitivity and negative predictive values of the 4T’s score were improved by considering positive or negative ELISA test results.

Conclusions: Consistent with previous reports, the PPV of a high probability 4T score alone was 100%. Thus, ELISA results might not change clinical decisions in this situation. Alternative anticoagulation might be inappropriately withheld in 20% of patients if a low probability 4T score alone were to be used for decision-making. The addition of ELISA data using an OD level of 0.4 increased the NPV to a threshold that might be considered clinically acceptable (94%) to withhold therapy. Addition of an OD level of ≥ 1 did not improve the PV of the 4 T’s. However, in contrast to other studies that found better predictive value to using this higher OD threshold, our study group was limited to people considered high risk for HIT. Validation of this strategy in a prospective trial with clinical endpoints should be considered.
Cause of Death among Patients with Local or Regional Stage Cancer of the Breast, Colon and Lung Who Develop Venous Thromboembolism (VTE).
Authors: Wun Ted , Chew Helen K. , Zhou Hong , Harvey Danielle , White Richard H.
Published: October 2019

Background: Previous data has shown that cancer-related VTE is associated with reduced two-year survival for patients with several tumor types. The reason for the survival difference is not clear.

Methods: Using the California cancer registry and hospital discharge data, we identified all cases diagnosed with colon, breast and lung cancer over a 6-year period. Patients with local or regional stage cancer who developed incident VTE within 2 years after the diagnosis of cancer were identified. The cause of death was determined using a master death registry. Each VTE case was compared to 4 case-controls without VTE after matching for age, race, sex, stage and number of medical co-morbidities. The odds ratio (OR) for dying from any cause and dying due to the underlying cancer in the subsequent 2 years was calculated.

Results (Table): Approximately 40% of all deaths occurred < 30 days of the VTE diagnosis for all cancers. The longer the time interval between cancer diagnosis and VTE, the greater the relative risk of dying of cancer.

Conclusions: Patients with lung, breast and colon cancer who developed VTE < 2 years after cancer diagnosis were significantly more likely to die, and death was attributed to the underlying cancer in a sizable percentage of these cases. This indicates that among patients initially diagnosed with local or regional stage cancer of the breast, colon or lung, VTE is frequently a harbinger or consequence of metastatic stage.

Odds Ratio (OR) for Death and Death Due to Cancer VTE Cases < 2 Years From Cancer Dx N Died < 2 Years From Date of VTE (%) OR (CI) For Death in VTE Cases < 2 Years Cancer As the Cause of Death N (%) OR (CI) for VTE Cases to Die of Cancer % Deaths Due to Cancer CI = Confidence Interval Local Stage     Breast 545 85 (16) 4.1 (3.0 – 5.6) 30 (6) 4.5 (2.7 – 7.7) 53     Colon 377 130 (35) 3.9 (3.0 – 5.1) 58 (15) 5.0 (3.3 – 7.5) 45     Lung 234 150 (64) 3.7 (2.6 – 5.5) 110 (45) 3.0 (2.0 – 4.4) 73 Regional Disease     Breast 493 116 (24) 3.4 (2.6 – 4.4) 73 (15) 2.8 (2.1 – 3.9) 63     Colon 887 375 (42) 2.1 (1.9 – 2.6) 273 (31) 2.1 (1.8 – 2.5) 73     Lung 485 386 (80) 3.7 (2.8 – 5.0) 320 (66) 2.5 (2.0 – 3.3) 66
PECAM-1 Inhibits Signaling Pathways That Amplify Platelet Aggregation.
Authors: Gao Cunji , Newman Peter J.
Published: October 2019

Though protein-tyrosine kinases get most of the attention, there is growing evidence that serine/threonine kinases (S/TKs) also play an important role in amplifying platelet activation responses following exposure to low-dose platelet agonists. For example, granule secretion is markedly impaired in platelets from mice deficient in either isoform of the STKs Akt1 (Blood104:1703, 2004) or Akt2 (J Clin Invest113:441, 2004), and pharmacological inhibition of the mitogen-activated protein kinase, p38, results in greatly reduced platelet aggregation and secretion (JBC271:6586, 1996; Blood107:965, 2006). Similarly, the integrin-linked kinase, ILK, is an S/TK that has recently been implicated in regulating both integrin activation (Thromb Haemost88:115, 2002; J Thromb Haemost2:1443, 2004) and cytoskeletal reorganization (Biochem J365:79, 2002; BBRC297:1324, 2002). We and others have previously shown that PECAM-1, a 130 kDa member of the immunoglobulin (Ig)-superfamily expressed on the surface of circulating platelets, leukocytes, and at the intercellular junctions of all continuous endothelium, functions to suppress platelet activation, though the mechanism by which this occurs has not been defined. To determine whether PECAM-1 might suppress S/TK-mediated platelet activation pathways, wild-type and PECAM-1 deficient murine platelets were stimulated with the GPVI-specific agonist, collagen-related peptide (CRP), and the activation state of Akt, p38, and ILK assessed biochemically. As previously reported, platelets from PECAM-1-deficient mice aggregated to a significantly greater extent in response to low-dose CRP than did their wild-type, PECAM-1-positive counterparts. Immunoblot analysis using phospho-specific antibodies revealed that both Akt and p38 existed in a hyper-activated state in CRP-stimulated, PECAM-1-deficient platelets - becoming phosphorylated both earlier and more intensively during platelet aggregation. ILK was similarly hyperactive, as assessed by determining the phosphorylation state of its substrate, glycogen synthase kinase 3β (GSK3β). To determine whether the inhibitory effects of PECAM-1 on S/TK-mediated platelet activation were dependant on prior fibrinogen binding to the major platelet integrin, GPIIb-IIIa, platelets were activated with low-dose CRP in the presence or absence of 2 mM RGDW. Pretreatment of platelets with RGDW had no effect on the ability of PECAM-1 to suppress activation of Akt,. p38, or ILK. Taken together, these data suggest that PECAM-1 functions as an inhibitory receptor in platelets, at least in part, by suppressing the activity of at least three different S/TKs, and thereby their ability to amplify early platelet activation responses.
Megakaryocyte-Derived Microparticles: Mechanism of Formation and Circulation in Plasma.
Authors: Flaumenhaft Robert , Dilks James R. , Patel Sunita R. , Klement Giannoula , Italiano Joseph E.
Published: October 2019

Platelet microparticles are submicron membrane vesicles expressing platelet markers and are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and increased platelet microparticle levels. Yet the physiology of these microparticles is poorly understood. Specifically, the origin of circulating platelet microparticles is not known. Platelet microparticles are routinely formed in vitro following exposure of platelets to pharmacologic concentrations of platelet agonists such as the combination of thrombin and collagen. This observation has lead to the widely held assumption that circulating platelet microparticles are derived from activated platelets. We now have identified and characterized platelet microparticles derived directly from megakaryocytes. Videomicroscopy of microparticle production by mouse megakaryocytes demonstrated an active process in which microparticles form as submicron beads along the lengths of thin, extended pseudopods. These pseudopods were morphologically distinct from the broad, branching cytoplasmic extensions from which proplatelets form. Studies using cytoskeletal inhibitors confirmed that megakaryocytes produce microparticles and platelets by two distinct mechanisms. The microtubule inhibitor nocodazole (5 μM) totally abolished proplatelet formation, but failed to affect microparticle formation. Latrunculin A (5 μM), which depolymerizes F-actin, inhibited proplatelet branching, but resulted in a 2-fold increase in megakaryocyte-derived microparticle formation. Megakaryocyte-derived microparticles had an average diameter of 0.5 μm. These microparticles were CD41+ and bound annexin V, indicated surface phosphatidylserine exposure. To determine whether plasma microparticles in wild-type mice are derived primarily from activated platelets or generated from megakaryocytes, we identified markers that distinguish between these two populations. CD62 and CD63 were found only on microparticles generated by activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not in microparticles from activated platelets. Circulating microparticles isolated from mice were CD62−,CD63−, and expressed full-length filamin A indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62−,CD63−, and expressed full-length filamin A. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles. We propose that circulating platelet microparticles can be derived from two sources. One source is the mature platelet, from which microparticles are generated following platelet activation in pathological conditions. The second source is megakaryocytes, which produce microparticles in a constitutive manner.
Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.
Authors: Jobe Shawn M. , Wilson Katina M. , Leo Lori , Molkentin Jeffery D. , Lentz Steven R. , Di Paola Jorge
Published: October 2019

Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A.

Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05).

Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses.

We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.
PKCδ Regulates Platelet Activity through the Inhibition of VASP-Mediated Filopodia Formation.
Authors: Pula Giordano , Schuh Kai , Walter Ulrich , Nakayama Keiichi I. , Nakayama Keiko , Poole Alastair W.
Published: October 2019

Filopodia are finger-like protrusions that play a crucial role in the guidance of cells through their extracellular and cellular environment. Blood platelets provide an essential early response system in haemostasis and arterial thrombosis and display a rapid and dramatic generation of filopodia upon activation, which play an essential role regulating their adhesive events leading to platelet aggregation. Here we report a novel and unexpected control mechanism for this explosive cellular rearrangement. Using pharmacological and genetic approaches we show that the novel isoform of protein kinase C, PKCδ, is a critical negative regulator of actin polymerization, filopodia formation and platelet aggregation. We show that PKCδ interacts with vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics, and regulates VASP phosphorylation. VASP is shown to be necessary for the PKCδ-dependent regulation of actin polymerization, filopodia formation and platelet aggregation. We therefore propose a causative link between the functional interaction of PKCδ with VASP and the role of the kinase as modulator of platelet activity.
Characterization of the Mechanism of Interaction between C-Reactive Protein and GPIIb/IIIa.
Authors: Brennan Marian P. , Moriarty Roisin D. , Arasu Stephanie , Foster Timothy J. , Cox Dermot
Published: October 2019

C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.
Charaterization of Human Primitive Megakaryocytes and “Platelets”.
Authors: Chen Dong , Lewis Rachel L. , Daigh Christine A. , Thomson James A.
Published: October 2019

Primitive megakaryocytes (PMs) have been first observed in murine embroynal yolk sac [Xu et al. Blood. 2001 Apr 1;97(7):2016–22.]. The biological characteristics and functional aspects of human primitive megakaryocytes are not well established even though they can be transiently observed by bone marrow stromal cell (OP9) co-culture methodology [Gaur, M et al J Thromb Haemost.2006 Feb;4(2):436–42.; Eto K et al. Proc Natl Acad Sci U S A.2002 Oct 1;99(20):12819–24.]. The goal of this study is to charaterize PMs’ unique biological and functional features. To obtain a pure population of human primitive megakaryocytes, human embryonic stem (hES) cells were cultured to form embryoid bodies.

CD34+/CD31+/VE-Cadherin+/Flk-1+ hemangioblasts on day 12 were isolated and further cultured for 10–12 days in media containing thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor (SCF). 30% of the non-adherent cells were strongly positive for CD31/CD41/CD61/CD42b, and weakly positive for CD42a/CD45/beta-2-microglobulin (B2M) but negative for CD117 or HLA-DR. The DNA ploidies are exclusively 2N–8N confirming the primitive status of PMs. Morphologically when compared to bone marrow derived megakaryocytes, PMs readily produce long processes with chains of “proplatelet” like structures. By live videomicroscopy, these “proplatelet” structures can actively recruit mitochondria. Interestingly, “platelet-like” fragment shedding from megakaryocytes is significantly stimulated in the presence of nitric oxide and human plasma. The “platelet-like” fragments and megakaryocytes respond to strong agonists, including thrombin and TRAP, with aggregation, alpha granule release and increased affinity for fibrinogen. In contrast, weaker agonists such as ADP only generate minimal response. This study provides preliminary evidence that primitive megakaryocytes have acquired some basic machinery of hemostasis even when the blood flow, liver function and coagulation factors are not present at the early phase of embryonal development. [We thank Dr.William L. Nichols at Mayo Clinic for his critical reviews of this abstract.]
Arrestin-2 Regulates Akt Activation by G Protein-Coupled Receptors in Platelets.
Authors: Li Dongjun , Woulfe Donna S.
Published: October 2019

Arrestins have been shown to play important roles in G Protein-Coupled Receptor (GPCR) function in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as scaffolds to recruit phosphatidyl inositol-3 kinases (PI3K)s to Gq-coupled receptors and promote PI3K-dependent signaling. Thrombin stimulates the PI3K-dependent activation of Akt in platelets in a Gq-dependent manner. Therefore, we sought to determine whether arrestins are involved in the PI3K-dependent activation of Akt in platelets. Comparative immunoblots show that of the two non-visual mammalian arrestins, only one, arrestin-2 (β-arrestin-1), is expressed in human and mouse platelets. Immunoprecipitation of arrestin-2 or p85-PI3K from platelet lysates demonstrated that arrestin-2 associates with the p85 subunit of PI3Ka/b in thrombin or ADP-stimulated platelets, but not resting cells. The association can be inhibited by inhibitors of the P2Y12 receptor for ADP, but not by P2Y1 inhibitors. p85-arrestin association is also blocked by inhibitors of src family kinases, as is Akt phosphorylation. To determine whether src family members were part of the p85-arrestin complexes, immunoblots were re-probed with antibodies to src, lyn and fyn. The results show that Lyn is incorporated into thrombin-stimulated arrestin complexes in a P2Y12-dependent manner. To determine whether arrestin-2 is important for Akt phosphorylation in platelets, megakaryocytes differentiated in culture from mouse embryonic stem cells were used as models of platelet signaling, since these cells are amenable to genetic manipulation. Arrestin-2 was inhibited in the cultured megakaryocytes using a siRNA approach, then cells were stimulated with thrombin and Akt phosphorylation was assessed by immunoblotting. Arrestin-2 expression in the cultured megakaryocytes treated with arrestin-2 specific siRNA was suppressed by an average of 53% compared to cells treated with scrambled siRNA, while thrombin-stimulated Akt phosphorylation was suppressed by 98% compared to scrambled siRNA-treated control cells (n=3 experiments, difference is significant, p=.01, unpaired student’s t-test). In conclusion, the results show that arrestin-2, lyn and PI3Kform a tri-molecular complex following stimulation of platelets with ADP or thrombin. Formation of arrestin complexes at activated receptor sites is important for the localized recruitment and src-dependent activation of p85-PI3K, thus promoting activation of Akt by G protein-coupled receptors.