Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1370-1370
Author(s):  
María Teresa Álvarez Román ◽  
Raul Justo Sanz ◽  
Elena Monzon Manzano ◽  
Monica Martín Salces ◽  
Ihosvany Fernandez Bello ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Compensatory Mechanism ◽  
Healthy Controls ◽  
Hepes Buffer ◽  
Prothrombinase Complex ◽  
Fibrinogen Receptor

Abstract Introduction: Immune thrombocytopenia (ITP) is an autoimmune disorder in which both increased platelet destruction and insufficient platelet production are involved. Patients can have a range of bleeding manifestations from none to severe at a similar platelet count. In some cases, patients have fewer bleeding symptoms than expected considering the low platelet count that they might have. Objective: The aim of this study was to determine the procoagulant profile of platelets from ITP patients in order to determine whether any of their features may explain this observation. Methods: Twenty-five patients with chronic ITP [(68±100)x109 platelets/L, mean age: 59.6 ± 16.1 years old, 56% female)] and thirty-five healthy controls [(256±36)x109 platelets/L, mean age: 41.6 ± 13.5 years old, 51% female) were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 152 g 10 min at 23°C for obtaining platelet rich plasma (PRP). To obtain washed platelets, the top two-thirds volumes of PRP were collected and centrifuged (650 g for 10 min at 23°C) after the addition of acid-citrate-dextrose (ACD, 1:10) and the pellet was resuspended in an equal volume of HEPES buffer. Platelet activation was determined by flow cytometry through binding of FITC-PAC1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 micromol/L thrombin receptor-activating peptide 6 (TRAP, Bachem, Switzerland) or 20 micromol/L ADP. Apoptosis was determined by flow cytometry analysis through FITC-annexin V binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions. To characterize platelet ability to bind coagulation factors, washed platelets (1x108/mL) were activated with 100 micromol/L TRAP and then incubated with FVa and/or FXa (5nM each, 10 min, ambient temperature). After fixation with 2% paraformaldehyde to cross-link the platelet-bound factors Va and Xa, platelets were washed two times with Hepes Buffer. Non-specific binding sites were blocked with 8% bovine serum albumin (30 min, room temperature). Following centrifugation, platelets were first incubated with anti-CD41-PE, anti-FVa and/or anti-FXa and then with a secondary FITC-goat anti-mouse IgG and stored at 4°C until flow cytometry analyses. Results: Platelets from ITP patients showed a basal expression of activated fibrinogen receptor similar to controls and a reduced ability for being activated by agonists (% of positive platelets for TRAP-induced PAC1 binding: 60±20 % in controls and 35±23 % in ITP, p<0.01; ADP-induced PAC1 binding: 63±14 % in controls and 50±23 % in ITP, p<0.05). Diminished responses to activation were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from ITP patients expressed more PS than controls under basal conditions [mean fluorescence (MF) for FITC-annexin V binding was: 336±128 in controls, 588±25 in ITP, p<0.05]. Since the PS is the anchor site of the prothrombinase complex, we studied the binding of FVa and FXa at baseline and after activating platelets with TRAP. The binding of these factors in both conditions was higher in the group of patients with ITP (MF for basal FVa binding: 41.4±14.4 in controls, 58.1±24 in ITP, p <0.02; MF for TRAP-induced FVa binding: 44.1±11.4 in controls, 81.4±38 in ITP, p<0.001; MF for basal FXa binding: 45.7±18.4 in controls, 58.1±24 in ITP, p <0.005; MF for TRAP-induced FXa binding: 46.1±16.4 in controls, 72.0±24 in ITP, p<0.05). The lower the platelet count the higher increase in PS exposure (Spearman r =-0,518, p <0.001) and the union of FVa (Spearman r = -0.8571, p <0.001) and FXa (Spearman r = -0.7455, p<0.05). Conclusions: Platelets from ITP patients, despite having less capacity of activation by agonist stimulation, have an increased procoagulant surface with greater ability to bind prothrombinase complex (FXaVa) than those from healthy controls. This feature might be a procoagulant compensatory mechanism that could reduce the risk of bleeding in patients with ITP. This work was supported by a grants from the FIS-FEDER, PI12/01831 and PI15/01457 Disclosures No relevant conflicts of interest to declare.

scholarly journals The Effect of the R882H DNMT3a Mutation on Megakaryocyte Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5186-5186
Author(s):  
Yang Lu ◽  
Dan Wang ◽  
Yifang Yuan ◽  
Li mei Chen ◽  
Yi ke Huang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Cell Lines ◽  
Colony Formation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Causal Relation ◽  
Meta Analysis ◽  
Annexin V

Abstract Introduction DNA methyltransferase (DNMT) family play an important role in the development and growth of lives, encoding enzymes that catalyze the addition of a methyl group to the cytosine residue of CpG islands. With the increase in methylation, the downstream genes are often associated with reduced expression. In this family, DNMT3a occupies the essential position to implement the de novo methylation. Timothy J. Ley and many other scientists found that in M4 and M5 acute myeloid leukemia (AML), around 20% patients suffered from DNMT3a mutation (most are R882H mutation), always associating with adverse prognosis. But what's the reason for adverse prognosis? Additionally, our formal Meta analysis showed that the de novo AML patients with DNMT3a mutation have higher platelet counts, WBC and RBC counts. To shed some light on the possible causal relation between the increasing in platelet count and poor prognosis led by DNMT3a mutation, we transduced the MK cell lines with genes null-mCherry (null), DNMT3a wild type-mCherry (DNMT3aWT) and DNMT3a R882H mutation type-mCherry (DNMT3aMT) respectively, trying to figure out the possible role that the mutation plays in the megakaryopoiesis and thrombopoiesis. Also, we tested several drugs that may target the mutation. Methods The SFFV-null-IRES-mCherry, SFFV-DNMT3aWT-IRES-mCherry and SFFV-DNMT3aMT-IRES-mCherry plasmids were constructed by Dr. Qianli Jiang, modified from LEGO-iC plasmids. MK cell lines (chrf-288-11, meg-01) were flow-through transduced with the lentivirus produced by packaging plasmids and those above. All the fluorescence positive cells have been doubly sorted by flow cytometry. Cell ploidy was analyzed by flow cytometry using Propidium Iodide (PI); colony forming unit (CFU-MK) were enumerated 14d after being plated with TPO and IL-3; cell proliferation were tested by CCK-8; apoptosis was measured via flow cytometry with PI and Annexin V-FITC; CD41a and CD61 were tested with flow cytometry. The drug tests including Decitabine, Dasatinib and Rituximab were analyzed using CCK-8 test and cytomorphologic tests. Results With CCK8 test of chrf-288-11 and meg-01, DNMT3aMT proliferates faster than the null and DNMT3aWT (P<0.05, Fig.1). In CFU-MK, both cells lines showed that DNMT3a mutation promoted the colony formation (P<0.05). The CD41a percentage decreased from null to DNMT3aWT and DNMT3aMT (P<0.05) while the CD61percentage increased from null to DNMT3aWT and DNMT3aMT (P<0.05). Also, with morphologic analyses, DNMT3aMT in both cell lines maintain more mature stages. Cell ploidy test also demonstrated that cell lines with DNMT3a mutation contain more multiploids (P<0.05). Apoptosis test illustrated that DNMT3a mutation protect the cell lines from apoptosis (P<0.05). In the drug experiments, 1uM Decitabine could slow down the proliferation of 3 gene types of chrf-288-11 significantly (P<0.05). Dasatinib also posed a negative effect on the proliferation of 3 gene types of chrf-288-11 (P<0.05, Fig.2). In Rituximab experiment, we could find that interestingly, certain concentrations could speed up the proliferation of 3 gene types of chrf-288-11, while others not (P<0.05, Fig.3). Conclusion With all the above evidences, we can safely conclude that the megakaryocyte cell lines with DNMT3a mutation are associated with high-differentiation, high-colony formation and low-apoptosis, which could help us to understand the elevation of platelet count in AML patients with DNMT3a mutation. The anti-apoptosis and renewal ability of the cell lines with DNMT3a mutation may lead to a bad prognosis of these AML patients (with DNMT3a mutation). What's more, according to the drug experiment, we found in the both cell lines, DNMT3aWT and DNMT3aMT cells died significantly at even low concentration of decitabine. Dasatinib also slowed down the proliferation of 3 gene types of chrf-288-11, whether Dasatinib could lead to further treatment of such leukemia with DNMT3A mutation needs more research. Rituximab is helpful in the treatment against refractory thrombocytopenia. However, the mechanism hasn't been clarified. Interestingly, our results showed that, Rituximab may have a direct effect on MKs, giving a boost to the megakaryopoiesis and thrombopoiesis with certain concentration. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Treatment of Primary Immune Thrombocytopenia with Thrombopoietin Receptor Agonists: Effect On Platelet Function and Plasma Thrombin Generation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1089-1089
Author(s):  
Ihosvany Fernández Bello ◽  
Victor Jimenez Yuste ◽  
Mayte Álvarez Román ◽  
Monica Martin Salces ◽  
Miguel Canales ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Platelet Function ◽  
Thrombin Generation ◽  
Annexin V ◽  
Normal Platelet Count ◽  
Fibrinogen Receptor ◽  
Normal Platelet ◽  
Thrombopoietin Receptor ◽  
Primary Immune Thrombocytopenia

Abstract Abstract 1089 Introduction: Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopenia caused by autoantibodies against platelet proteins that lead to platelets destruction and suboptimal platelet production. Platelet count and bleeding phenotype vary widely between ITP patients. In spite of the low platelet number, some thrombocytopenic ITP patients seldom bleed which might indicate the presence of additional mechanisms that contribute to the haemostasis in such patients. Objective: Thrombopoietin receptor agonists (TPO-RA) have recently been introduced for the treatment of patients with ITP and can noticeably increase platelet count in most of the ITP patients, even in those with severe refractory thrombocytopenia. The aim of this work was to study platelet function and thrombin generation in thrombocytopenic ITP patients before treatment with TPO-RA and once they had recovered the normal platelet count as consequence of the treatment. Methods: Fourteen ITP patients with thrombocytopenia (TP-ITP, platelet count less than 100,000 platelets/microliter) were studied before starting TPO-RA treatment (4 patients with romiplostim and 10 patients with eltrombopag) and after reaching a platelet count higher than 100,000 platelets/microliter (NP-ITP). Thirty-three healthy subjects were included as the control group. Platelet activation was determined by flow cytometry through binding of FITC-PAC-1 (a mAb that recognizes activated conformation of fibrinogen receptor) to quiescent and 100 microM TRAP activated platelets. Immature platelet fraction was determined labeling platelets with thiazole orange. Expression of fibrinogen receptor was determined by flow cytometry with specific mAbs. Apoptosis was determined by flow cytometry through FITC-annexin V binding to phosphatidylserine (PS). Thrombin generation was measured in platelet-free plasma by the method of Hemker (Calibrated Automated Thrombography, CAT). Activation was performed with a final concentration of 4 microM of phospholipids and 1 pM of tissue factor. Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Values of p≤0.05 were considered statistically significant. Results: Platelets from TP- and NP-ITP patients showed a basal expression of activated fibrinogen receptor similar to controls. Platelets from TP-ITP patients presented a reduced ability for being activated by TRAP (binding of PAC-1 expressed as % of positive platelets: 89±19 % in controls and 56±22 % in TP-ITP, p<0.01). TPO-RA treatment did not improve platelet activation despite increasing platelet count (binding of PAC-1: 47±19 % in NP-ITP, p<0.01). Diminished responses to TRAP were not due to a reduction in surface expression of fibrinogen receptor in platelets from ITP patients. Platelets from either TP- or NP-ITP patients, expressed more PS than controls under basal conditions. Percentage of platelets that bound FITC-annexin V were: 47±12 % in controls, 63±13 % in TP-ITP and 60±15 % in NP-ITP, p<0.001). Percentage of immature platelets in TP-ITP patients were higher than in controls (0.8±0.9 % in controls and 6.6 ±4.0 % in TP-ITP, p<0.05) and was reduced by TPO-RA treatment (in NP-ITP patients: 2.2 ±1.7 %). CAT experiments showed that TP-ITP patients had an increased endogenous thrombin potential (ETP, p<0.001) and reached higher maximum levels of thrombin (peak height, p <0.001) than controls. This procoagulant characteristic of plasma from TP-ITP patients was unchanged after treatment with TPO-RA and the recovery of a normal platelet count. Conclusions: Our results showed that treatment of ITP patients with TPO-RA is effective for increasing platelet production but did not ameliorate platelet function or procoagulant conditions of the disease which may indicate that treatment with TPO-RA do not interfere with the mechanisms involved in impairment of platelet function and generation of a procoagulant state. This increased thrombin generation of plasma from ITP patients has to be taken under consideration when evaluating thrombotic risk of therapeutic treatments, and we would like to point out the need of performing more studies to elucidate causes of the increased thrombogenic potential observed in ITP patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluation of Platelet Function Defects in Patients with Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1021-1021
Author(s):  
Elena Monzón Manzano ◽  
María Teresa Alvarez Román ◽  
Andres Ramirez Lopez ◽  
Elena G Arias-Salgado ◽  
Paula Acuña ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Platelet Function ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Healthy Controls ◽  
Activation Markers ◽  
Platelet Production ◽  
Neuraminidase Activity ◽  
Fibrinogen Receptor

Abstract Background: Primary immune thrombocytopenia (ITP) is a megakaryocytic (MK)/platelet-specific autoimmune disorder characterized by platelet count &lt;100×10 9/L with or without bleeding manifestations, and diagnosed by exclusion of other causes of thrombocytopenia. It is widely accepted the involvement of platelet autoantibodies on deterioration of platelets from patients with ITP. Moreover, an enhanced activity of neuraminidase may also reduce sialic acid from glycoside residues on platelet surface, especially from the highly glycosylated von Willebrand factor (vWF) receptor. Because controversial results regarding the functionality of platelets from ITP patients can be found in literature, we aimed to determine platelet ability to be stimulated by agonists. Moreover, we aimed to determine the way anti-platelet auto- antibodies (abs) and neuraminidase activity may affect the function of platelets derived from MKs of healthy controls. Methods: This observational, prospective and transversal study included 42 patients with chronic primary ITP and 55 healthy controls. Platelet fibrinogen and vWF receptors and activation markers (PAC1 binding to activated fibrinogen receptor and exposure of P-selectin after agonists treatment), were evaluated by flow cytometry. Presence of Antibodies (abs) against platelet's glycoproteins in ITP serum was analysed with a Luminex based assay (LifecodesPak Lx). Neuraminidase (NEU) activity in serum was determined with the substrate 20-(4-methylumbelliferyl)-a-D-N-(MUNANA). Human CD34 + cell-enriched population was obtained with CliniMACS (MiltenyiBiotec) from G-CSF mobilized peripheral blood of a healthy donor. For MK differentiation, CD34 + cells were cultured 12 days in StemSpan™ Serum-Free Expansion Medium II (SFEM II) with 50ng/ml of recombinant human thrompoietin. Then, 10% of serum from healthy controls (4) or ITP patients (4) were added to the culture of mature MKs and incubated for 3 days. Phenotypic analysis of MKs and culture derived-platelets was carried out using abs against CD34, CD41, CD42a and CD42b.Platelet-like particles were considered as CD41-positive events with a size (FSC) and granularity (SSC) scatter properties similar to blood platelets. Culture-derived platelets were stimulated with 100 µM TRAP and 10 µM ADP and activation markers were analyzed by flow cytometry. Results: Expression of fibrinogen receptor on platelets from ITP patients were similar to those from healthy controls but showed a reduced capacity to be activated. Impairment in platelet degranulation measured as exposition of P-selectin after agonist's stimulation was also observed in platelets from these patients (Figure 1). Of note, surface content of CD42b subunit of vWF receptor was reduced (Figure 1). To determine whether diminished platelet function might be due to a plasma component, we induced platelet production from MK of healthy controls as referred in Methods. Abs against platelets and neuraminidase activity were determined in serum samples. Serum from 4 healthy controls or from 4 ITP patients (1 with anti-CD42b, 1 with anti-GPIa-IIa and 2 with undetectable abs) were added to MKs culture. No differences existed in MK differentiation and platelet production between MKs incubated with serum from healthy controls or from ITP patients, but similarly as observed in platelets from ITP patients, MK-derived platelets had an impaired ability to be activated (Table 1). Platelets derived from MKs incubated with ITP serum with anti-platelet abs had also a diminished exposure of CD42b (73±8% of controls). Moreover, neuraminidase content of these samples was slightly higher than that from ITP samples without abs (130 vs 100 % of controls). Conclusion: Platelets from ITP patients had a diminished ability to be stimulated. In vitro study showed that megakaryopoiesis was normal in presence of ITP serum, but released platelets had a lower ability to be activated. Involvement of abs in this effect cannot be ruled out despite we detected abs only in 2 of the tested sera because efficiency of method to detect these abs is ~ 50%. On the other hand, reduced levels of CD42b might be due to the increased activity of neuraminidase. Reduction of sialic acid from CD42b might initiate its metalloproteinase-mediated cleavage or change affinity of the ab used for its detection. Research funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Alvarez Román: Pfizer: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding. García Barcenilla: Roche: Speakers Bureau; Takeda: Speakers Bureau; Bayer: Speakers Bureau; SOBI: Speakers Bureau. Canales: Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria; Gilead/Kite: Consultancy, Honoraria; Eusa Pharma: Consultancy, Honoraria; Incyte: Consultancy; Karyopharm: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau. Jiménez-Yuste: Grifols: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding. Butta: Novo-Nordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Roche: Speakers Bureau; CSL-Behring: Research Funding.

scholarly journals Platelet Features from ITP Patients Responders to Different Therapeutic Treatments

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4648-4648
Author(s):  
Nora Butta ◽  
María Isabel Rivas Pollmar ◽  
María Teresa Álvarez Román ◽  
Monica Martín Salces ◽  
Ihosvany Fernandez Bello ◽  
...  
Keyword(s):  
Platelet Count ◽  
Plasma Levels ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Procoagulant Activity ◽  
Control Group ◽  
Severe Bleeding ◽  
Healthy Controls ◽  
Platelet Counts ◽  
Before And After

Abstract Background: Patients with ITP have a wide variation in the presentation of the disease, platelet count and their clinical course. The decision to begin treatment is based on the hemorrhagic symptoms and platelet count. Intravenous immunoglobulin (IVIG) is usually associated with glucocorticoid administration in patients with severe bleeding or platelet counts <20x109/L and a quick response is required. Agonists of thrombopoietin receptor (TPO-AR) and splenectomy are other therapeutic tools for these patients. Materials and Methods: We recruited patients with ITP before and after responding to treatment with IVIG (n = 11) and AR-TPO (4 patients with romiplostim and 10 with eltrombopag), 5 splenectomized patients and 82 healthy controls. The percentage of reticulated platelets, platelet activation and binding of annexin-V were evaluated by flow cytometry. Plasma levels of TPO and "a proliferation-inducing ligand" (APRIL) were determined by ELISA. Procoagulant activity associated microparticles (MP) and the ability of plasma to generate thrombin were determined, respectively, with Zymuphen kit and calibrated automated thrombinography (CAT) triggered by 1 pM tissue factor and 4 micromolar phospholipid (PPP-low reagent, Diagnostica Stago, Spain). Results: Patients with ITP that respond to IGIV and AR-TPO treatments recovered platelet counts without reaching the levels of the control group, whereas the platelet count in splenectomized patients did not differ from it. Plasma levels of TPO and the number of immature platelets in the first two groups were higher than in controls before responding to treatment. Despite recovering platelet count, platelet capacity of being activated by agonists such as TRAP (thrombin receptor agonist for PAR-1) was less than that of the controls in all groups. This decrease was not due to a reduction in the expression of the fibrinogen receptor on platelets from ITP patients. Platelets from ITP patients before and after responding to all treatments studied, showed more phosphatidylserine exposure and greater microparticles-associated and plasma-associated procoagulant activity. Plasma levels of APRIL, a factor that stimulates B cells and antibody production, decreased in ITP patients who responded to the AR-TPO, reaching the levels observed in the control group. In the group of splenectomized patients a decrease of APRIL was also observed, but still remained higher than in healthy controls. Conclusions: ITP patients who respond to treatment with IVIG and AR-TPO and undergoing splenectomy recovered platelet count but not its function. The treatments did not modify the microparticles- and plasma-associated thrombogenic capacity. Among all the treatments studied, AR-TPO and splenectomy had an addittional benefical effect reducing APRIL plasma levels Disclosures No relevant conflicts of interest to declare.

Administration of Anti-Platelet Antibodies Prevents the Anti-Platelet Immune Response and Bleeding Complications of Neonatal Immune Thrombocytopenia in a Murine Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 223-223
Author(s):  
Heidi Tiller ◽  
Pingguo Chen ◽  
Bjorn Skogen ◽  
Mette Kjaer Killie ◽  
Anne Husebekk ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Platelet Count ◽  
Murine Model ◽  
Immune Thrombocytopenia ◽  
Human Platelet ◽  
Bleeding Complications ◽  
Platelet Antibodies ◽  
Β3 Integrin ◽  
Equity Ownership

Abstract Abstract 223 Background: The human platelet antigen (HPA) 1a is a potent immunogen located on the β3 integrin. Ten % of pregnant HPA1a negative women produce antibodies against the HPA1a antigen if the foetus is HPA1a positive. Fetal/neonatal immune thrombocytopenia (FNIT) can occur if the mother develops alloantibodies against fetal platelets, with intracranial haemorrhage as the most severe complication. The current opinion has been that immunization against the HPA1a antigen takes place during the first HPA1 non-compatible pregnancy. However, results from a large and recent screening study in Norway found that the majority (75%) of women were immunized around time of delivery, and not so often during pregnancy. This indicates that FNIT could be more similar to haemolytic disease of the newborn (HDN) than previously thought. To prevent HDN, antibody mediated immune suppression (AMIS) is induced by administration of anti-D antibodies in connection with RhD-negative pregnancies. The same principle could be used to prevent FNIT by administration of anti-HPA1a antibodies in HPA1a-negative pregnancies. We have previously established a murine model of FNIT using β3 integrin-deficient (β3−/−) mice. The first aim of the current project was to test whether administration of human anti-HPA1a IgG could suppress the anti-human platelet immune response in β3−/− mice after transfusion of human HPA1a positive platelets. For the second part of the project, we used a pure murine model to test whether administration of murine anti-β3 antibodies transfused after delivery could induce AMIS and prevent bleeding complications of FNIT in the subsequent pregnancies. Methods: Human/murine model: Human IgG from 5 donors with high levels of anti-HPA1a antibodies was purified by Protein G affinity chromatography. Purified IgG from one male donor without detectable anti-platelet specific antibodies was used as control IgG. Human platelets were isolated from an HPA1a positive donor. β3−/− mice were immunized by one tail vein transfusion with 2 × 106 human HPA1a positive platelets, with or without subsequent transfusion of 900ug human IgG (100% saturation). After 7 days, the mice were bled and sera collected. The anti-human platelet immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG as detection antibody. Six mice were injected with anti-HPA1a containing IgG. Control IgG (n=6) or no IgG (n=4) were used as negative controls. Pure murine model: High-titer anti-β3 sera were produced by 4 weekly transfusions of 108 wild type (WT) platelets to β3−/− mice. Naïve β3−/− female mice were bred with naïve β3−/− male mice. Within 24 hours of delivery, the mother was transfused with 108 WT platelets with or without immediate transfusion of anti-β3 sera. The transfusions were repeated one week after delivery and the same females were bred again with WT male BALB/c mice. The anti-β3 immune response was analyzed via flow cytometry, using FITC-conjugated goat anti-mouse IgG. The FNIT phenotype was monitored and all live pups were bled from the carotid vein to determine platelet count. Results: Administration of purified anti-HPA1a IgG significantly suppressed the anti-human platelet immune response in β3−/− mice after transfusion of HPA1a positive platelets as compared with control IgG (p < 0.05). In the pure murine model of FNIT, the anti-β3 immune response was markedly suppressed during the subsequent pregnancy in the mice treated with anti-β3 sera. Two out of three mice receiving anti-β3 sera treatment delivered live pups with moderate thrombocytopenia without signs of haemorrhage (mean platelet count 217 ×106/mL). The third mouse receiving anti-β3 sera delivered dead pups. In contrast, all female mice (n = 3) without anti-β3 sera treatment miscarried. Conclusions: We have demonstrated in vivo that AMIS can be induced by administration of anti-platelet antibodies using a murine model of FNIT. Preliminary data indicates that bleeding complications of FNIT can be prevented with this prophylactic approach. Disclosures: Skogen: Prophylix Pharma a/s: Employment, Equity Ownership. Killie:Prophylix Pharma a/s: Equity Ownership. Husebekk:Prophylix Pharma a/s: Equity Ownership. Kjeldsen-Kragh:Prophylix Pharma a/s: Equity Ownership.

Lactadherin as a Probe for Phosphatidylserine Exposure and as An Anticoagulant for the Procoagulant Activity in the Study of Stored Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3150-3150
Author(s):  
Jin Zhou ◽  
Jinxiao Hou ◽  
Wen Li ◽  
Xiaoqian Zhang ◽  
Yueyue Fu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Progressive Increase ◽  
Procoagulant Activity ◽  
Platelet Concentrates ◽  
Time Analysis ◽  
Coagulation Time ◽  
Enzymatic Assays

Abstract Abstract 3150 Poster Board III-87 Background Phosphatidylserine (PS) can support coagulant reactions. However, it is uncertain how the location and extent of PS exposure to the membranes of stored platelets affect such reactions. We compared annexin V with lactadherin as a way of detecting how of PS exposure influences the procoagulant properties of stored platelets in platelet concentrates (PCs). Method PS exposure and the relevant procoagulant activity (PCA) of platelets in 5 different PCs were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. PS exposure was separately measured using annexin V and lactadherin, respectively. Results Exposure of PS to stored platelets promoted thrombin formation. A progressive increase in PS exposure was detected by flow cytometry. Moreover, using lactadherin, we identifed higher levels of PS exposure on the platelets and platelet-derived microparticles (PMPs) compared to detection using annexin V. The percentage of PS-positive cells was 0.02 % by annexin V versus 0.3 % by lactadherin on day 0, 7.5 % by annexin V versus 12.3 % by lactadherin on day 5, and 29 % by annexin V versus 44.3 % by lactadherin on day 9. Rare microparticles (MPs) were released from fresh platelets, and, the number of PMPs increased approximately 2-fold on day 5 and further progressively increased. Using lactadherin and platelets in the earlier stage of storage, confocal microscopy revealed earlier and localized PS exposure based on plasma membrane staining. For later storage platelets, increased levels of PS-positive platelets and PMPs were clearly detected by both annexin V and lactadherin. Thirty-two nM lactadherin or annexin V prolonged coagulation time 2.4 fold versus 2 fold. The productions of thrombin and intrinsic/extrinsic factor Xase were approximately inhibited 85 % and 60 % by lactadherin and annexin V, respectively. Conclusion PS exposure was localized to the cellular rims, blebbing vesicles and thin elongated filopodia-like areas on banked platelets. Furthermore, lactadherin provides a more accurate measurement of PS exposure and the relevant with PCA, which is an important factor to consider for transfusion medicine. Our findings of elevated PS-positive platelets and PMPs indicate that platelets should not be stored for extended periods of time. Disclosures No relevant conflicts of interest to declare.

Dynamic Changes in Platelet Responsiveness to Thrombin and Coated Platelet Potential May Inform Risk of Hemorrhage and/or Thrombosis in a Novel Canine Mode of Immune Thrombocytopenia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2193-2193
Author(s):  
Marshall A. Mazepa ◽  
Dana N LeVine ◽  
Adam J Birkenheuer ◽  
Marjory B Brooks ◽  
Shila K Nordone ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Count ◽  
Exploratory Study ◽  
Immune Thrombocytopenia ◽  
Treated Group ◽  
Canine Model ◽  
Severe Thrombocytopenia ◽  
Dynamic Changes ◽  
Platelet Recovery ◽  
Time Zero

Abstract Abstract 2193 In both canine and human patients with Immune Thrombocytopenia (ITP), bleeding risk is challenging to predict, and potentially leads to over-treatment of patients at low risk. Conversely, recent studies have highlighted the risk of thrombosis in ITP during platelet recovery. Given these clinical observations, we hypothesized that in ITP, changes in platelet response to agonists may occur in addition to changes in platelet numbers. In response to dual agonist activation (thrombin and convulxin), a subpopulation of platelets in both humans and dogs develops enhanced procoagulant activity. This subpopulation is termed coated platelets, and differences in individuals' potential to form coated platelets have been correlated with both hemorrhagic and thrombotic outcomes. In this exploratory study, we serially evaluated ex vivo platelet responsiveness to both thrombin and dual agonists (termed coated platelet potential) in a novel canine model of ITP. Dogs (n=4) were infused with a murine monoclonal anti-GPIIb antibody (2F9) in order to model ITP and generate predictable severe thrombocytopenia. Control dogs (n=3) were infused with a control antibody. Platelet count, thrombin responsiveness, and coated platelet potential were measured at baseline, time zero, 6 hours, 24 hours, and every 24hrs thereafter until the platelet count was ≥ baseline for at least two consecutive measures (recovery). Time zero was defined as the time when platelet count first fell to ≤ 30,000/μl following 2F9 infusion, or 1 hour following control antibody infusion. For platelet thrombin responsiveness, a monoclonal antibody to P-selectin was used to determine platelet P-selectin surface expression by flow cytometry after stimulation with graded doses of thrombin. The ED50 Thrombin was defined as the concentration of thrombin required for half-maximal P-selectin expression. Coated platelet potential was defined as the percent of platelets activated to the highly procoagulant state after dual stimulation with thrombin and convulxin, as determined by binding of biotinylated fibrinogen by platelets by flow cytometry. All dogs in the treated group developed severe thrombocytopenia (median=6×103, range=4–11×103 platelets/uL); no dogs in the control group developed thrombocytopenia. All treated dogs had platelet recovery by 240 hours (median=132 hours, range 120–240hours). Of interest, at 6 hours, ED50 Thrombin in the treated group increased nearly twofold (fig 1A) (ratio of median ED50 Thrombin treated/baseline=1.6, range 1.3–2.3), which correlated with a decline in coated platelet potential by nearly half of baseline (fig 1B) (median 52.4% of baseline, range 19.6–61.5%); minimal change from baseline was observed in controls. In both groups, ED50 Thrombin was lower at recovery than baseline (fig 1A) (treated median ED50 Thrombin=71.5% of baseline; control median ED50 Thrombin=67% of baseline). A trend of rising coated platelet potential was also noted as platelets recovered in the treated group. In conclusion, in this exploratory study of a canine model of ITP, we observed dynamic changes in platelet responsiveness. During severe thrombocytopenia, we observed a rise in ED50, indicating a decline in response to thrombin, which correlated with a fall in coated platelet potential. We speculate that this early fall in platelet thrombin response and coated platelet potential could contribute to hemorrhage risk in ITP. As a complement to this finding, in the treated group, there was a rise in coated platelet potential as platelets rebounded and coated platelet potential was slightly greater than baseline at recovery. This is consistent with others' observation that younger platelets are more likely to have coated platelet potential. We also observed a decline in ED50 Thrombin at recovery, not only in the treated dogs, but also control dogs. Thus, at recovery, the decline in ED50 Thrombin was independent of treatment group. However, this may be an artifact of our small sample size. Our observed increase in coated platelet potential during platelet recovery could potentially contribute to the thrombotic tendency of some ITP patients. Future studies are planned to explore the relationship of hemorrhagic and thrombotic risk with platelet thrombin responsiveness and coated platelet potential in this model of ITP and clinical studies of canine and human ITP. Disclosures: No relevant conflicts of interest to declare.

The Ratio of Treg/Th17 Cells Correlates with the Platelet Number in Different Disease State of Primary Immune Thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4649-4649
Author(s):  
Lili Ji ◽  
Feng Li ◽  
Yanxia Zhan ◽  
Fanli Hua ◽  
Shanhua Zou ◽  
...  
Keyword(s):  
Autoimmune Diseases ◽  
Th17 Cells ◽  
Immune Thrombocytopenia ◽  
Conflicts Of Interest ◽  
Disease Onset ◽  
Treg Cells ◽  
Healthy Controls ◽  
T Helper ◽  
Platelet Number ◽  
Primary Immune Thrombocytopenia

Abstract Abstract 4649 Background: Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4+T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Tregs to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio. Methods: Thirty adult patients with newly diagnosed ITP enrolled in this study. Patients who needed treatment had been clinically followed up for 12 months. The percentages of CD4+CD25hiFoxp3+ Treg cells and CD3+CD4+IL-17-producing Th17 cells in these patients and healthy controls (n=17) were longitudinally analyzed by flow cytometry. Results: The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls and the percentage of Th17 cells increased significantly at disease onset. It is suggested that the ratio of Treg/Th17 correlated with the disease activity. Conclusion: The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients. Disclosures: No relevant conflicts of interest to declare.

Preclinical Development Of a Nanomedicne Approach For Multiple Myeloma Targeting The Myc Oncoprotein

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4228-4228
Author(s):  
Deepti Soodgupta ◽  
Dipanjan Pan ◽  
Grace Hu ◽  
Angana Senpan ◽  
Xiaoxia Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Delivery ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Free Drug ◽  
Preclinical Development ◽  
Late Antigen ◽  
Mtt Assays

Abstract Purpose This study investigated alpha 4 beta 1/ Very Late Antigen-4 (α4β1/ VLA-4)-integrin targeted nanotherapy approach to deliver a new lipase-labile prodrug. Experimental Design A phospholipid-based MYC-MAX inhibitor prodrug (MI1-PD) was synthesized, and its inherent anti-proliferate potency was compared to the lipid-free compound (MI1) using mouse multiple myeloma (MM) cell line (5TGM1). VLA-4-targeted perfluorocarbon (PFC) nanoparticles binding to 5TGM1 cells was measured and compared to biomarker expression assessed with flow cytometry using antibodies. The efficacy of MI1-PD incorporated into non-targeted and VLA-4-targeted PFC NP exposed to 5TGM1 cells was assessed with MTT assays, Annexin V and cell cycle analysis. Results MI1-PD was more potent by several orders of magnitude than its free drug counterpart in culture. Targeted NP binding correlated well with biomarker expression assessment by flow cytometry in 5TGM1 cells. Non-targeted NPs had no appreciable binding to 5TGM1 cells. High anti-MM potency of MI1-PD was noted in VLA-4-targeted NPs compared to the non-targeted NPs demonstrating that the efficacy was dependent on expression of the targeted biomarker to afford particle-to-cell drug delivery. Conclusions These results suggest the feasibility of an improved integrin VLA-4- targeted nanotherapy approach to deliver a lipase- labile prodrug construct, MI1-PD. Disclosures: No relevant conflicts of interest to declare.

Combined Inhibition of Wee1 and Poly(ADP Ribose) Polymerase1/2 Synergistically Inhibits Acute Myeloid Leukemia Cells By Enhancing DNA Damage and the Induction of Apoptosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3621-3621 ◽  
Author(s):  
Jonathan C Snedeker ◽  
Tamara M Burleson ◽  
Raoul Tibes ◽  
Christopher C. Porter
Keyword(s):  
Flow Cytometry ◽  
Cell Proliferation ◽  
Dna Damage ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Combination Index ◽  
Annexin V ◽  
Concomitant Treatment ◽  
Western Blots

Abstract Introduction: Successful treatment of AML remains dependent upon cytotoxic chemotherapy. However, traditional regimens are not well tolerated by older patients who are at highest risk of disease, and salvage rates after relapse are low, necessitating novel therapeutic strategies. Our groups identified Wee1 as a potential therapeutic target in AML, particularly in the context of concomitant treatment with cytarabine (Tibes et al, Blood, 2012; Porter et al, Leukemia, 2012). Wee1 inhibits CDK1&2 via phosphorylation thereby stalling cell cycle progression. One consequence of Wee1 inhibition/CDK1 activation is impairment of DNA repair via homologous recombination (Krajewska et al, Oncogene, 2013). Cells in which HR is impaired are dependent upon Parp1/2 function, and HR deficient cells are particularly sensitive to Parp1/2 inhibition. Therefore, we hypothesized that combined Wee1 and Parp1/2 inhibition may result in greater inhibition of AML cell proliferation and survival than either alone. Methods: Human AML cell lines, MV4-11 and Molm-13, and a mouse AML that expresses MLL-ENL/FLT3-ITD were cultured with various concentrations of a Wee1 inhibitor (AZ1775) and a Parp1/2 inhibitor (olaparib) and counted 72 hours later by propidium iodide exclusion and flow cytometry. In some experiments, cells were split into fresh media to recover for 72 more hours. Combination Index (CI) values were calculated by the method of Chou and Talalay. Apoptosis was measured using Annexin V/7AAD and flow cytometry. Western blots were used to confirm inhibition of CDK1/2 phosphorylation and to measure DNA damage induction (gamma-H2AX). Results: Combined inhibition of Wee1 and Parp1/2 was synergistic, as measured by cell numbers at 72 hours, in all 3 cell lines tested, with combination index values ranging from 0.3 to 0.9. When cells were allowed to recover after treatment, those treated by single agents were able to continue proliferating. However, those treated with the combination did not recover as well or at all, indicating greatly impaired proliferative capacity. Combined inhibition of Wee1 and Parp1/2 also resulted in a significant increase in apoptosis greater than either drug alone. Western blots for gamma-H2AX confirmed that the combination of Wee1 and Parp1/2 resulted in more DNA damage than either drug alone. Discussion: Combined inhibition of Wee1 and Parp1/2 results in greater inhibition of AML cell proliferation, DNA damage and apoptosis than either drug alone. Future studies will include experiments with primary patient samples, as well as in vivo trials combining Wee1 inhibition with Parp1/2 inhibition. These preliminary studies raise the possibility of rational combinations of targeted agents for leukemia in those for whom conventional chemotherapeutics may not be well tolerated. Disclosures No relevant conflicts of interest to declare.

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