Platelet Features from ITP Patients Responders to Different Therapeutic Treatments

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.

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Defective Functional Activity of Platelets in Children with Severe Chronic Immune Thrombocytopenia and Its Correction By Romiplostim Treatment

Blood ◽

10.1182/blood.v124.21.1466.1466 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1466-1466

Author(s):

Irina Demina ◽

Elena Suntsova ◽

Alexey Maschan ◽

Michael Maschan ◽

Galina Novichkova ◽

...

Keyword(s):

Platelet Count ◽

Functional Activity ◽

Conflicts Of Interest ◽

Annexin V ◽

Dense Granule ◽

Control Group ◽

Platelet Counts ◽

Chronic Itp ◽

Increased Risk ◽

Granule Release

Abstract BACKGROUND: Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction, which together with suboptimal platelet production lead to thrombocytopenia. Children with low platelet counts due to ITP are at an increased risk of bleeding, which is not always directly correlated to the severity of thrombocytopenia. Relationship of platelet functional activity and bleeding risk in ITP is poorly investigated. There are conflicting reports on whether platelets in adult ITP patients differ from normal ones, and no data for children are available. Information about the thrombopoietin receptor agonists (eltrombopag and romiplostim, which are increasingly used to treat ITP) effect on platelet quality is also scarce. Data for eltrombopag are inconclusive, and no studies for romiplostim are known. OBJECTIVES: To investigate platelet functional activity before and during the romiplostim treatment of ITP in children. METHODS: The study group consisted of patients from 4 to 11 years old (n=8) with chronic ITP; the control group included healthy donors (n=12). All patients initially had low platelet counts (<15*109/L) and did not respond to the first- and second- line therapy. Blood samples were collected before treatment with romiplastim and then monthly until the onset of clinical remission. Platelets in whole blood were either left intact or activated with collagen-related peptide (0.18 mkg/ml) and thrombin receptor activating peptide (12.5 mkM), labelled and analysed by flow cytometry. To characterize platelet functions, we used fluorescencently labeled antibodies against glycoprotein Ib (CD42b), total and active integrin αIIbβ3 (CD61 and PAC-1), and P-selectin (CD62P); dense granule release was assessed using platelet loading with mepacrine, and procoagulant activity was determined with a phosphatidylserine marker annexin V. RESULTS: All investigated parameters of platelet function in children with severe chronic ITP before treatment were greatly impaired. CD42b and CD61 were up to several-fold lower the normal values, while phosphatidylserine exposure, dense granule release and integrin activation upon activation were decreased by at least an order of magnitude. Romiplostim treatment improved platelet parameters, although not always to the normal level. Five patients out of eight responded partially or completely to the romiplostim therapy. The clinical response (relief of hemorrhagic manifestations) correlated well with improvement of the functional state of platelets, in one case, even without significant platelet count increase. CONCLUSIONS. Our data suggest essential revision of the pathophysiology of severe chronic ITP in children: their platelets are not only small in their number, but also appear to have severe defects of all major functions. The mechanism of action of romiplostim in these patients might also be in some need of revision, as it seems to greatly improve not only quantity, but also quality of platelets. Our data demonstrate the important role of monitoring platelet functional activity in addition to platelet count. Disclosures No relevant conflicts of interest to declare.

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Circulating Microparticles and Risk of Venous Thromboembolism.

Blood ◽

10.1182/blood.v114.22.3987.3987 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3987-3987

Author(s):

Paolo Bucciarelli ◽

Emanuele Previtali ◽

Ida Martinelli ◽

Andrea Artoni ◽

Serena M Passamonti ◽

...

Keyword(s):

Body Mass Index ◽

Venous Thromboembolism ◽

Body Mass ◽

Plasma Levels ◽

Conflicts Of Interest ◽

Factor V Leiden ◽

Control Group ◽

Dependent Manner ◽

Healthy Controls ◽

Increased Risk

Abstract Abstract 3987 Poster Board III-923 Background Microparticles (MPs) are circulating, submicroscopic fragments (<1 μm of diameter) of membrane-bound cytoplasm that shed from the surface of an activated or apoptotic cell and play a role in coagulation, inflammation, cell remodelling and proliferation. There is increasing evidence that MPs are involved in thrombosis, but whether or not they are an independent risk factor for venous thromboembolism (VTE) is not established. Aim of the study To investigate the association between high plasma levels of MPs and risk of VTE Patients and Methods In a case-control study, 186 patients with a first episode of VTE (deep venous thrombosis and/or pulmonary embolism) and 418 healthy controls were included. MPs were analyzed by flow cytometry with a gate defined by a 1 μm beads and using APC-Annexin V together with FITC anti-CD41 or FITC anti-CD142 antibodies in order to identify platelet MPs (MP-Plts) and MPs exposing tissue factor (MP-TF), respectively. MPs levels were expressed as number/μL. Results Patients had significantly higher median plasma levels of both MPs-Plts and MPs-TF than controls [1942 vs 1519 (p<0.0001) and 579 vs 454 (p<0.0001)]. Higher median levels of MP-Plts and MP-TF were found in 41 patients who underwent blood sampling within 6 months from VTE than in those sampled later [2114 vs 1694 (p=0.086) and 652 vs 543 (p=0.120)]. Sex, age, body mass index and factor VIII plasma levels had no influence on MPs levels, as well as the use of oral contraceptives (this latter evaluated only in controls). In the whole study population, carriership of thrombophilia (antithrombin, protein C or protein S deficiency, factor V Leiden, prothrombin G20210A, antiphospholipid antibodies, hyperhomocysteinemia or combined abnormalities) had higher levels of MP-Plts and MP-TF than non-carriers [1907 vs 1565 (p=0.002) and 532 vs 468 (p=0.011)]. The odds ratio (OR) for VTE, adjusted for sex, age, body mass index and thrombophilia was 2.5-fold higher in individuals with MPs plasma levels >95th percentile of the control group (3633/μL for MPs-Plts and 1113/μL for MPs-TF) than in those with MPs levels ≤95th percentile [for MPs-Plts: OR=2.59 (95%CI 1.23 – 5.45); for MPs-TF: OR=2.38 (1.15 – 4.92)]. The risk increased in a dose-dependent manner for both MPs-Plts and MPs-TF, particularly above the 75th percentile of the distribution in controls. The exclusion of patients whose MPs levels were measured within 6 months from VTE (in order to avoid the possible effect of the acute phase on MPs measurements), did not change the results [adjusted OR: 2.63 (1.18 – 5.89) for MPs-Plts and 2.36 (1.10 – 5.19) for MPs-TF]. The Table shows the relative risks of VTE associated with the presence or absence of high MPs levels and thrombophilia. Individuals with MPs >95th percentile or thrombophilia alone had a 2 to 3-fold increased risk of VTE, whereas those with both MPs-Plts >95th percentile and thrombophilia had a 9-fold increased risk of VTE. This synergistic effect was confirmed also for MPs-TF and remained after the exclusion of patients whose blood sample was collected within 6 months from VTE [OR 7.72 (1.68-35.4) for MP-Plts and 8.14 (2.08-31.8) for MP-TF]. Conclusions Plasma levels of MPs are significantly higher in patients with VTE than in healthy controls. MPs levels >95th percentile are associated with a 2.5-fold increased risk of VTE. There is a synergistic interaction between high levels of MPs and thrombophilia on VTE risk. Disclosures: No relevant conflicts of interest to declare.

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Increased Microparticle-Linked Procoagulant Activity In Patients with Primary Immune Thrombocytopenia.

Blood ◽

10.1182/blood.v116.21.3707.3707 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3707-3707

Author(s):

Elena G. Arias Salgado ◽

Ihosvany Fernández Bello ◽

Mayte Álvarez Román ◽

Isabel Rivas ◽

Mónica Martín Salces ◽

...

Keyword(s):

Platelet Count ◽

Immune Thrombocytopenia ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Lag Time ◽

Peak Height ◽

Procoagulant Activity ◽

Control Group ◽

Increased Risk ◽

Primary Immune Thrombocytopenia

Abstract Abstract 3707 Primary immune thrombocytopenia (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia (platelet count less than 100,000/μL) and the absence of any obvious initiating and/or underlying cause for the thrombocytopenia. In spite of the low platelet number, some thrombocytopenic patients seldom bleed, indicating the existence of other factors that regulate haemostasis in these patients. Elevated levels of plasma microparticles (MPs) had been observed in IPT patients. MPs are vesicles with a size less than 0.5 micrometers, derived from cell membranes after their activation or apoptosis. Most MPs are highly procoagulant, expressing annexin V binding sites and tissue factor. However, relatively little is known of their specific functions in ITP. In the present study we aim to elucidate if a relationship exists between microparticle-linked procoagulant activity and haemostasis in ITP patients. Twenty-two ITP patients, 3 male and 19 female, aged between 25 to 92 years, were included. Sixteen age- and sex-matched healthy individuals were used as control group. Platelet-related primary haemostasis was evaluated with an automated platelet function analyzer (PFA-100®, Siemens Healthcare Diagnostics). Samples of citrated blood were aspirated under a shear rate of 4,000–5,000/s through a 150-micrometer aperture cut into a collagen-ADP (COL-ADP) or collagen-epinephrine (COL-EPI) coated membrane. The platelet haemostatic capacity is indicated by the time required for the platelet plug to occlude the aperture (closure time), which is expressed in seconds. MP procoagulant activity was determined with ZYMUPHEN MP-Activity kit (Aniara, Mason, Ohio) and by calibrated automated thrombography (CAT) in plasma samples obtained after 2 centrifugations at room temperature (first: 15 min at 1,500 g, second: 2 min at 13,000 g). These methods measure endogenous thrombin generation. CAT evaluates four parameters of thrombin generation: the endogenous thrombin potential (ETP), lag time, time to peak (TTP) and peak height. PFA-100® determinations with COL-EPI and COL-ADP cartridges in blood samples from ITP patients with less than 50,000/μL showed longer closure times than control group (p<0.05), whereas samples from ITP patients with a platelet count between 50,000/μL and 100,000/μL showed closure times of the same order of magnitude as control ones (platelet count ranging from 162,000 to 368,000)μL).Plasma from these patients had higher MP-mediated procoagulant activity evaluated with ZYMUPHEN kit (control 6.1+3.9 nM, ITP group 10,1±8.2 nM, p<0.05) as well as with CAT (ETP (nM*min): control: 1692.6±341.9, ITP: 2191,8±398.9, p<0.01; lag time (min): control: 19.9±8.2, ITP: 14.3±4.3, p<0.05; TTP (min): control: 22.0±8.3, ITP:16.3±4.4, p<0.05; peak height (nM): control: 389.7±70.6, ITP: 498,8±97.5, p<0.01). Our results indicate that increased MP procoagulant activity in ITP patients may be protective against bleeding events that should be observed in those thrombocytopenic conditions. Three of the ITP patients included in this study had been splenectomyced and we consider of interest to point out that two of them in spite of recovering a normal platelet count still maintain a high MP procoagulant activity. This observation agrees with a recent work that postulates that MPs might contribute to an increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy (Fontana et al, Thromb Research, 2008;122:59). Disclosures: No relevant conflicts of interest to declare.

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Tissue Factor-Dependent Pro-Coagulant Activity Of Human Platelets Is Directly Related To Membrane Cholesterol Content. Rosuvastatin, But Not Atorvastatin, Reduces The Platelet Cholesterol, Tissue Factor Protein and Clotting Activity In Hypercholesterolemic Patients

Blood ◽

10.1182/blood.v122.21.34.34 ◽

2013 ◽

Vol 122 (21) ◽

pp. 34-34

Author(s):

Olga Panes ◽

Juan P. Valderas ◽

Mónica Acevedo ◽

Susana Contreras ◽

Jaime Pereira ◽

...

Keyword(s):

Tissue Factor ◽

Plasma Levels ◽

Conflicts Of Interest ◽

Daily Intake ◽

Fold Increase ◽

Procoagulant Activity ◽

Serotonin Secretion ◽

Before And After ◽

Induced Aggregation

Abstract Plasma levels of LDL-cholesterol (LDL-Cho), a major risk factor of atherothrombosis, is in equilibrium with platelet(Plt) membrane Cho. In hypercholesterolemia (Hypercho) the membrane ratio Cho/phospholipids is increased and the platelets (Plts) seem to be hyper responsive to agonists, generating more TxA2. Plts are not only involved in the pathogenesis of atheromatosis, but also are key players in thrombotic events. This supports the benefit of primary/secondary prevention by decreasing the plasma LDL-Cho with statins and the Plt function with inhibitors of Plt aggregation/secretion. However, the Tissue Factor (TF)-induced procoagulant contribution of activated Plts has been largely ignored. Aims to study the relationship between Plt TF activity and membrane Cho, both in vitro and in 45 patients with Hypercho (LDL-Cho>140 mg dL-1), age and sex-matched with 37 healthy controls (Ctrl) with LDL-Cho<130 mg dL-1. We also studied the effect of 1-month daily intake of either 40 mg atorvastatin (Atorv, n=21) or 20 mg rosuvastatin (Rosuv, n=24). In in vitro studies, membrane Cho increased by 31% in normal Plts after incubation with LDL-Cho (p=0.031, n=5). This was associated with 1.57-fold increase in ADP-induced aggregation (AUC, p=0.0012, n=16) and no increase in serotonin secretion. Increase in collagen-induced aggregation was marginal (4%), though significant (p=0.026, n=16), and 5-HT secretion increased by 22% (p=0.0002). Plt TF-induced generation of FXa was similar in Plts with and without LDL-Cho incubation in basal conditions. However, Plt activation with VWF-Ris increased Plt FXa generation from 134 (in Ctrls) to 205 nmol 2*10-7plts (p=0.04, n=12). Ctrl and patients (before and after statins) did not differ in age (49 year-old), BUN, glucose, and plasma levels of HDL-Cho, CK, usCRP, TNFa, IL-6, IL-8, PAI-1 and TAT complexes. Plasma Cho fell similarly after Atorv and Rosuv to 87 and 81 mg dL-1, whereas fibrinogen levels, similar in patients and Ctrls before the intervention, increased 13% after Atorv and 3% after Rosuv (p=0.01). Plt membrane Cho in Ctrl and Hypercho patients were 91 and 137 μg mg-1 protein (p=0.005), respectively. Platelet Cho did not change significantly after Atorv (168μg mg-1), but fell to 71μg mg-1 protein after Rosuv (p=0.0053), a value even lower than Ctrl (p=0.055). In contrast with in vitro Cho enrichment of platelet membranes, patients with Hypercho and Ctrl have similar PRP aggregation/secretion with arachidonate, ADP, epinephrine, collagen and ristocetin, both before and after statins. TF protein in Plt membranes (ELISA) was similar in Ctrl and patients with Hypercho (485±123 vs 1133±166 pg mg-1 protein, SEM, p=0.014). No change in platelet TF was observed with Atorv, but a non-significant fall from 1254 to 798 pg mg-1 protein was observed after Rosuv (798±149). Basal FXa generation of resting Plts was very low and similar for Ctrl and Hypercho, before and after statins. After stimulation with VWF-Ris, Hypercho Plts generated more FXa than Ctrl (Median 139[Range 11-3990]) vs 46[7-493] nmol 2 *10-7 Plts) (p=0.0056). Atorv did not decrease VWF-Ris-induced FXa generation. In contrast, the increased FXa generation induced by VWF-Ris in Plts from patients treated with Rosuv was similar to that of stimulated Ctrl Plts (46[7-493] vs 54[7-701] nmol FXa 2*10-7Plt). Conclusions Our results indicate: 1. Patients with Hypercho do not have increased Plt aggregation-secretion, and these functions are not affected by statin therapy. 2. Plts acquire Cho on incubation with LDL-Cho, and patients with Hypercho have increased membrane Cho. 3. Plt membrane Cho is normalized by treatment with Rosuv, but not with Atorv. 4. Hypercho patients have increased platelet TF membrane protein, with a tendency to decrease after Rosuv, but not after Atorv. 5. Higher Plt TF-induced generation of FXa is observed in patients with Hypercho than in Ctrl after Plt activation. 6. Atorv treatment does not result in Plt FXa generation decrease, but FXa measured after Rosuv is similar to that of Ctrl. 7. In summary, these results strongly suggest that a major role of Hypercho in atherothrombosis is the increased platelet TF-dependent procoagulant activity, and that this effect is better controlled by Rosuv than by Atorv. This would constitute another pleiotropic effect of Rosuv. 8. Reduction of Plt procoagulant activity would be a novel target in prevention/treatment of atherothrombosis. Disclosures: No relevant conflicts of interest to declare.

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Metabolomic Profile in Patients with Paroxysmal Nocturnal Hemoglobinuria

Blood ◽

10.1182/blood-2019-126241 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2229-2229

Author(s):

Patricia Eiko Yamakawa ◽

Ana Rita Da Fonseca ◽

Matheus Vescovi ◽

Iara Baldim Rabelo Gomes ◽

Ismael Dale Cotrim Guerreiro da Silva ◽

...

Keyword(s):

Mass Spectrometry ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Conflicts Of Interest ◽

Signs And Symptoms ◽

Control Group ◽

Operational Characteristic ◽

Healthy Controls ◽

Metabolomic Profile ◽

Before And After ◽

Functional Measure

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a non-malignant clonal disease of hematopoietic cells with a complex pathophysiology and variable clinical spectrum, characterized by signs and symptoms related to intravascular hemolysis, hypercoagulability, and cytopenias. Metaboloma is the end product of an organism's genetic configuration plus the influence of all factors to which it is exposed. Metabolomic profile is able to provide a more accurate functional measure of a phenotype formed by the result of genomic, transcriptomic and proteomic changes. Aims: To compare a metabolomic profile in the PNH hemolytic group with healthy controls, and to compare a metabolomic profile before and after the administration of eculizumab in PNH patients. Methods: To perform metabolomic profile, we used mass spectrometry in 23 patients with hemolytic PNH and in 166 healthy adults, as control group. Twelve PNH patients samples were also collected before and 24 hours after receiving eculizumab. Liquid chromatography with mass spectrometry was performed using the AbsoluteIDQ P180 Biocrates kit (Biocrates, Life Science AG, Innsbruck, Austria): 186 metabolites from 7 different classes were identified and quantified. The data were imported to the analytical site MetaboAnalyst 4.0 and ROCCET: ROC Curve Explorer & Tester for the generation of Univariate and Multivariate Operational Characteristic curves of the Receiver (ROC). Results: In the patients with hemolytic PNH, of the 186 metabolites analyzed, 92 of them showed significant differences between patients and controls, with positive or negative correlation. The major metabolites increased in the PNH group were long-chain acylcarnitines, while the major metabolites reduced in the PNH group were histidine, taurine, glutamate, glutamine, aspartate and phosphatidylcholines. The C14:1/C16:1 ratio lower than 1.4 was shown to be a reliable marker in patients with PNH, suggesting a disorder in lipid elongation. Besides, PNH patients had a significant increase in C6:1 levels and C14:1/C6 and C4/C6 ratios. After receiving eculizumab, patients reached levels comparable to those of normal subject. Conclusion: We observed differences in the metabolome of PNH patients as compared to healthy controls, suggesting a unique metabolomic profile of these patients, characterized by an altered acylcarnitine balance, reduction in aminoacids participating in the glycogenesis pathway and an impaired glutaminolysis. Acylcarnitines levels seem to significantly reduce with the use of eculizumab, demonstrating that the use of the antibody has action in reducing hemolysis and possibly in mitochondrial function of these patients. Disclosures No relevant conflicts of interest to declare.

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Effects Of Thrombopoietin Receptor Agonists On APRIL Plasma Levels In Patients With Immune Thrombocytopenia

Blood ◽

10.1182/blood.v122.21.1083.1083 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1083-1083

Author(s):

Nora V. Butta ◽

Mayte Álvarez Román ◽

Ihosvany Fernández Bello ◽

Elena G. Arias Salgado ◽

Isabel Rivas Pollmar ◽

...

Keyword(s):

Platelet Count ◽

T Cell ◽

Regulatory T Cell ◽

Plasma Levels ◽

Immune Thrombocytopenia ◽

Cell Activity ◽

Healthy Controls ◽

Platelet Counts ◽

Chronic Itp ◽

Thrombopoietin Receptor

Abstract Introduction Immune thrombocytopenia (ITP) is an example of an autoimmune disease in which B-lymphocytes produce autoantibodies against platelets. Antibody-mediated platelet destruction and suboptimal platelet production leads to a decrease in platelet count. ITP patients with thrombocytopaenia have increased plasma levels of a proliferation-inducing ligand (APRIL), a factor that can promote B-cell maturation and survival. Two new compounds that bind to the thrombopoietin receptor (TPO-R) and activate the megakaryopoiesis have been recently approved for the treatment of chronic ITP as second-line treatment. Objective It has been recently reported an improved regulatory T-cell activity in patients with chronic ITP treated with TPO-R agonists (TPO-RA) (Bao et al, 2010). So we aimed to evaluate the effect of TPO-RA treatment on APRIL plasma levels in ITP patients before (ITP-1) and after responding (ITP-2) to the treatment. Methods This was an observational and prospective study. Thirteen patients with chronic ITP in whom treatment with a TPO-RA was indicated, and thirty-three healthy controls were included. ITP patients were studied at two times: at inclusion (ITP-1), when platelet count was less than 30x109/L for patients without concomitant medication or less than 65x109/L for patients receiving corticosteroids or intravenous immunoglobulin; and after a response to TPO-RA therapy was elicited (ITP-2). The response to TPO-RA was defined as a platelet count >30x109/L in patients without additional treatment or >65x109/L for those with concomitant treatments. EDTA-anticoagulated whole blood was centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma which was then centrifuged at 10,000 g for 15 min at room temperature. Supernatant plasma was stored at –70°C until analysis. Plasma TPO and APRIL concentrations were determined using a commercially available enzyme-linked immunosorbent assay (ELISA, Duoset-R&D, Minneapolis, Mn, USA). Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Comparisons of quantitative variables were made with ANOVA and Dunn test. Results were expressed as mean±SD. Correlations were calculated with Spearman test. Values of p≤0.05 were considered statistically significant. Results Platelet count in the ITP-1 group ((23±17)x109/L) increased after responding to TPO-RA to values similar to controls (controls: (233±77)x109/L and ITP-2: (140±36)x109/L). TPO plasma level was higher in ITP-1 patients (30.05±26.81 pg/ml, p<0.005) than in healthy controls (7.36±11.74 pg/ml) but not significantly different when compared with the values of the ITP-2 group (26.81±17.62 pg/ml). ITP-1 patients showed significantly higher APRIL plasma levels (37.60+28.73 ng/ml, p<0.0001) than controls (2.20±3.11 ng/ml) and ITP-2 patients (4.92+4.68 ng/ml), indicating that TPO-RA treatment caused a diminution in APRIL plasma levels. When looking into the relationship between APRIL plasma levels and platelet count, a significant correlation was only found in the ITP-1 group (r=-0.5919, p<0.05). This supports the potential role of APRIL in the reduction of platelet counts in ITP patients. Conclusion ITP patients with thrombocytopaenia that responded to TPO-RA treatment increased their platelet count reducing plasma APRIL levels and without changing the moderately high levels of plasma TPO. Reductions in APRIL levels caused by TPO-RA treatment could be an additional mechanism that contributes to an increased platelet count in ITP patients treated with these agents. Bao W, Bussel JB, Heck S, et al. Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood. 2010 Nov 25;116(22):4639-4645 Disclosures: No relevant conflicts of interest to declare.

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Thromboelastographic Follow-Up Of Prophylactic Thrombocyte Transfusion

Blood ◽

10.1182/blood.v122.21.4826.4826 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4826-4826 ◽

Cited By ~ 1

Author(s):

Deniz Goren Sahin ◽

Olga M. Akay ◽

Mustafa Karagulle ◽

Eren Gunduz ◽

Zafer Gulbas

Keyword(s):

Platelet Count ◽

Economic Burden ◽

Platelet Transfusion ◽

Conflicts Of Interest ◽

Bleeding Risk ◽

Thrombocyte Count ◽

Platelet Counts ◽

Before And After ◽

Thrombocyte Transfusion

Introduction Prophylactic thrombocyte transfusion is being used to reduce increased bleeding risk after chemotherapy treatment for leukemia or malignancy. This transfusion is frequently applied when thrombocyte count is below <10.000/uL or between 10.000 and 20.000/uL. However, it was shown that thrombocyte count alone is not enough for determining bleeding risk. Moreover, given the fact that thrombocyte transfusions have inherent risks and economic burden, new laboratory approaches such as thromboelastography can be considered to determine bleeding risk in these patients. Thromboelastography is a new alternative method to conventional coagulation tests, which gives information about hemostatic system by evaluating clot’s visco-elastic and mechanical features. The aim of our study to establish a transfusion algorithm by thromboelastographic follow-up of prophylactic thrombocyte transfusion. Methods Eighty patients who have been diagnosed as acute leukemia were randomized into 4 groups. Six units random thrombocyte was given to the first group, three units random thrombocyte was given to the second group, one unit apheresis was given to the third group, and ½ unit apheresis was given to the fourth group. Before and 15 minutes after transfusion, peripheral blood was taken and CBC and rotation thromboelastograpy (ROTEM) was performed by standard device (Pentapharm GmbH, Munich, Germany). Clotting time (CT), clot formation time (CFT), and maximum clot firmness (MCF) were evaluated by 2 methods, in-TEM and ex-TEM. Patients were followed up during study by using clinical bleeding signs based on WHO bleeding grade. Patients who used medications that can affect thrombocyte functions within the last 14 days and patients who have systemic disorders (renal, hepatic, endocrinological) or hemostatic disorders were not included in this study. Variance analysis was used in order to find out statistical differences. P<0.05 was considered statistically significant. Results When platelet counts and ROTEM results were analyzed for each parameter before platelet transfusion, there were no statistically significant differences among groups. We analyzed the differences of platelet counts and thromboelastographic parameters before and after prophylactic platelet transfusion and we didn’t see any statistically significant differences between groups. Clinical bleeding signs were not correlated with platelet count in any groups. Conclusion Six units random, three units random, complete apheresis or half apheresis prophylactic platelet transfusion does not cause any significant changes in platelet count, ROTEM parameters and clinical bleeding signs. Therefore, low dose platelet transfusion can be considered because of its lower economic burden. Moreover, further studies are needed to evaluate the potential of ROTEM as an independent factor for transfusion indication. Disclosures: No relevant conflicts of interest to declare.

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Procoagulant Profile of Platelets from Immune Thrombocytopenia Patients

Blood ◽

10.1182/blood.v128.22.1370.1370 ◽

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.

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Changes in Platelet Aggregability after Ovariectomy

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657029 ◽

1979 ◽

Vol 42 (04) ◽

pp. 1332-1339 ◽

Cited By ~ 7

Author(s):

Hiroh Yamazaki ◽

Takeshi Motomiya ◽

Minoru Sonoda ◽

Noboru Miyagawa

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Ovarian Hormones ◽

Control Group ◽

Platelet Aggregability ◽

Appearance Rate ◽

Before And After ◽

The Difference ◽

Induced Aggregation ◽

Observation Period

SummaryChanges in platelets in 48 patients with uterine myoma before and after hysterectomy with and without ovariectomy were examined. Bilateral ovariectomy in 25 cases (ovariec-tomized group) and unilateral or non-ovariectomy in 23 cases (control group) were performed at the hysterectomy. Platelet count and an appearance rate of secondary aggregation decreased at one day after and increased at one week after the operation, similarly in both the ovariectomized and the control group. The appearance rate of secondary aggregation was reflected in an intensity of aggregation at 5 min after the addition of reagent to PRP. At one month after the operation, the appearance rate of secondary aggregation induced by 3 μM ADP showed a statistically significant decrease in comparison with the preoperation value (P <0.05) and the enhancement of 5-min aggregation was still observed in the control group, while ceased in the ovariectomized group. The difference between the two groups was significant (P < 0.05). There was almost no change in the speed and intensity of primary and secondary aggregation during the observation period. No significant differences in collagen-induced aggregation were noted between the two groups. The results suggest that ovarian hormones, mainly estrogen, facilitate platelet activation which is mediated by the so-called secondary aggregation.

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Analysis of complete blood counts in rotaviral gastroenteritis with special emphasis on platelet indices

International Journal of Contemporary Pediatrics ◽

10.18203/2349-3291.ijcp20192756 ◽

2019 ◽

Vol 6 (4) ◽

pp. 1567

Author(s):

Mahesh B. Maralihalli ◽

Kavan R. Deshpande ◽

Pallavi K Deshpande

Keyword(s):

Platelet Count ◽

Acute Phase ◽

Acute Phase Reactant ◽

Healthy Controls ◽

Distribution Width ◽

Platelet Counts ◽

Rotavirus Gastroenteritis ◽

Phase Reactant ◽

Significant Difference ◽

Complete Blood Counts

Background: The objectives of this study was to analyze complete blood counts in rotaviral gastroenteritis with special emphasis on platelet indices.Methods: Children diagnosed as rotavirus gastroenteritis and healthy controls were enrolled in this study. Severity of acute gastroenteritis was classified into mild, moderate and severe grades using Vesikari score. Rotavirus was determined in fresh stool samples using rapid diagnostic rotavirus antigen test. Hemoglobin, leukocyte, neutrophil to lymphocyte percentage ratio, platelet counts, mean platelet volume (MPV), platelet distribution width (PDW) and platelet crit (PCT) levels were assessed for all children. It’s a case control study conducted at Pediatric Speciality Hospital.Results: There were 30 cases with mean age 1.58 years. Healthy controls were 30 with mean age 2.10 years. Mean Hb was lower in cases. Mean of platelet counts was higher in cases. Mean MPV levels was lower in cases. Mean PCT value was higher in cases. Mean MPV to platelet ratio value was lower in cases. All parameters values showed no significant difference among mild, moderate and severe groups of rotaviral gastroenteritis cases. Platelet count was negatively correlated with Hb, MPVP and positively correlated with TLC and PCT. MPV was positively correlated with MPVP and PDW. PCT was negatively correlated with Hb, MPVP and positively correlated with TLC and platelet count.Conclusions: MPV can be used as negative acute phase reactant in rotavirus gastroenteritis and so is the MPV to platelet ratio. Platelet count is acute phase reactant in rotavirus gastroenteritis and so is the platelet crit value.

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