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

Blood ◽  
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.

Microparticle-Associated Thrombogenic Mechanism Might Compensate Bleeding Tendency in Patients with Myelodysplastic Syndromes.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2821-2821
Author(s):  
Nora V. Butta ◽  
Monica Martin Salces ◽  
Raquel de Paz ◽  
Ihosvany Fernández Bello ◽  
Mayte Álvarez Román ◽  
...  
Keyword(s):  
Platelet Count ◽  
Myelodysplastic Syndromes ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Time Lag ◽  
Procoagulant Activity ◽  
Bleeding Tendency ◽  
Compensatory Mechanisms ◽  
Increased Risk ◽  
Spearman Test

Abstract Abstract 2821 Introduction: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders with clonal bone marrow anomalies characterized by ineffective hematopoiesis, morphologic and functional hematopoietic cells abnormalities, and an increased risk of transformation to acute myeloid leukemia. Previous work from our lab has shown that MDS patients had an impairment in platelet activation, more externalization of phosphatidylserine (PS) indicating an increased platelet apoptosis and, in some cases, thrombocytopenia. However, these patients do not bleed at the frequency that would be expected taking into account these facts. Objective: The aim of this work was to identify the existence of some compensatory mechanisms that could be counteracting the expected bleeding tendency in these patients. Since apoptosis is accompanied by membrane blebbing and production of PS-exposing procoagulant microparticles (MP) shed from the plasma membrane, we assessed the thrombogenic capacity of MP and evaluate their correlation with exposure of PS. We also studied the ability of plasma to generate thrombin after stimulus. Methods: Seventy-five patients with MDS, 36% female, mean age 77 years (range: 41 to 93 years) and sixty-eight healthy controls, 29% female, mean age 52 years (range 20 to 76 years) were included. Whole blood was collected in citrated tubes and centrifuged at 1,500 g for 15 min at 23°C to obtain platelet poor plasma (PPP). PPP was subjected to 2 additional centrifugations at room temperature (first: 15 min at 1,500 g and second: 2 min at 13,000 g) following SSC-ISTH recommendations to obtain platelet free plasma (PFP). MP procoagulant activity was determined in PFP with the ZYMUPHEN MP-Activity kit (HYPHEN BioMed, Neuville sur Oise, France). Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) as described by Hemker at final concentration of 1pM tissue factor and 4 microM phospholipids. Four CAT parameters were recorded: endogenous thrombin potential (ETP, total amount of thrombin generated over time), lag time (time to the beginning of the explosive burst of thrombin generation), time to peak (TTP, time to reach the maximum thrombin concentration) and peak height (maximum thrombin concentration obtained). Apoptosis was determined by assessing the level of PS externalization by the binding of FITC-Annexin V to platelets by flow cytometry. Thrombocytopenia was defined as platelet count less than 50,000 platelets/microliter. Results: Platelet count in MDS patients varied from 4,000 to 478,000 platelets/microliter. We observed an increased MP-associated procoagulant activity in MDS patients with thrombocytopenia compared to controls (p<0.05). This would be related to the increased apoptosis observed in platelets from MDS patients with thrombocytopenia (Spearman test r = 0.655, p <0.01). CAT evaluation of plasma procoagulant activity did not show differences between patients and controls in any of the measured parameters. Conclusions: Our results showed an increased procoagulant activity associated to plasma MPs in MDS with thrombocytopenia that might contribute to the minor risk of bleeding observed in these patients. However, further studies are necessary to assess the clinical significance of this finding. Disclosures: No relevant conflicts of interest to declare.

Clinical Determinants of Thrombin Generation Measured in Presence and Absence of Platelets—Results from the Gutenberg Health Study

2018 ◽  
Vol 118 (05) ◽  
pp. 873-882 ◽  
Author(s):  
Andreas Schulz ◽  
Henri Spronk ◽  
Aline Beicht ◽  
Dagmar Laubert-Reh ◽  
Rene van Oerle ◽  
...  
Keyword(s):  
Platelet Count ◽  
Thrombin Generation ◽  
Linear Regression Analysis ◽  
Lag Time ◽  
Peak Height ◽  
Health Study ◽  
Individual Risk ◽  
Dependent Manner ◽  
Important Indicator ◽  
Gutenberg Health Study

AbstractThe tendency of a plasma sample to generate thrombin, a central enzyme in blood coagulation, might be an important indicator of prothrombotic risk linked to cardiovascular disease (CVD), but the presence of platelets may be a critical determinant. Clinical data, laboratory markers and thrombin generation (TG), investigated in both platelet-rich plasma (PRP) and platelet-free plasma (PFP) at 1 pM TF, were available in 407 individuals from the Gutenberg Health Study. Given the well-known effect of anticoagulants on TG, subjects taking anticoagulants (n = 15) have been excluded resulting in 392 subjects for further analysis. Lag time, endogenous thrombin potential (ETP) and peak height were the investigated parameters of a TG curve. Multivariable linear regression analysis was used to identify TG determinants. Mean platelet volume (MPV) and platelet count were both negatively associated to lag time and positively to peak height (MPV, β:6.35 [2.66; 10.0]; platelet count, β:0.111 [0.054; 0.169]) in PRP only. C-reactive protein was positively associated with lag time and ETP in both PRP and PFP, with a stronger effect on ETP in PRP (PRP, β:76.7 [47.5; 106]; PFP, β:34.8 [10.3; 59.2]). After adjustment for fibrinogen, the relation between CRP and ETP was attenuated in PRP and PFP. Of the traditional cardiovascular risk factors (CVRFs), obesity was positively associated to TG in PRP only. Our findings support that TG, particularly in PRP, relates to traditional CVRFs in a representative sample from a population-based study. Assessment of procoagulant activity in a platelet-dependent manner by TG is a promising tool for assessing individual risk for CVD.

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

Blood ◽  
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.

Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1763-1763
Author(s):  
Wenche Jy ◽  
Jaehoon Bang ◽  
Loreta Bidot ◽  
Andrew Lin ◽  
Joaquin J. Jimenez ◽  
...  
Keyword(s):  
High Speed ◽  
Thrombin Generation ◽  
Calcium Ionophore ◽  
Lag Time ◽  
Peak Height ◽  
Calcium Ionophore A23187 ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Differential Centrifugation

Abstract BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation. METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb.2002;32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF (&lt;0.1%) but very high AnV binding (88±6%). By high speed centrifugation (15,000xg for 10 min), two populations of RMP were studied: the larger RMP in the pellet expressed GlyP, AnV and Ulex while the smaller or lighter RMP remaining in the supernatant, did not express GlyP and AnV but do express Ulex. The smaller RMP accounted for 30–40% of total Ulex+ RMP. These two subspecies (large and small) of RMP showed distinct thrombin generation profiles. The lag time and peak height of thrombin generation for large RMP (GlyP+/AnV+/Ulex+) was 23–28 min and 300–335 nM, respectively, which is close to values of whole RMP. On the other hand, the smaller RMP (Ulex+/GlyP−/AnV−) produced much longer lag time (31–38 min) and lower peak (60–75 nM), indicating that the majority of the procoagulant activity of RMP is associated with larger RMP. SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.

Comparison of the Use of TFPI Inhibition and Plasma Dilution to Increase the Sensitivity of Thrombin Generation Assay to Measure the Procoagulant Activity of Microvesicles

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4392-4392
Author(s):  
Damien DG Gheldof ◽  
François Mullier ◽  
Bernard Chatelain ◽  
jean-Michel Dogne ◽  
Christian Chatelain
Keyword(s):  
Venous Thromboembolism ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Surrogate Marker ◽  
Predictive Biomarker ◽  
Procoagulant Activity ◽  
Assay Sensitivity ◽  
Thrombin Generation Assay ◽  
Increased Risk

Abstract Abstract 4392 Introduction: Patients with cancer have a 7- to 10- fold increased risk of developing venous thromboembolism. Circulating microvesicles (MVs) could be a predictive biomarker for venous thromboembolism in cancer. Thrombin generation assay is a useful technique to detect procoagulant activity of MVs. However, thrombin generation assay suffers from a lack of sensitivity due to the presence of Tissue Factor Pathway Inhibitor (TFPI) in plasma. Aims: To improve the sensitivity of thrombin generation assay to tissue factor (TF) by limiting the interference of TFPI. Methods: Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate MVs. Samples were then centrifuged and supernatants which contain MVs were used for thrombin generation assay. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of thrombin generation assay to detect procoagulant activity of MVs. Results: i) Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intra-assay variability. ii) Plasma dilution had no impact on the thrombin generation assay sensitivity when thrombin generation assay was triggered by MVs derived from MDA-MB-231. Conclusions: Thrombin generation is a very sensitive method to study the procoagulant activity of TF-MVs. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz Domain I. A twice plasma dilution is an interesting alternative to study the procoagulant activity of MVs by thrombin generation assay with a good sensitivity, especially when low plasma quantities are available. Disclosures: No relevant conflicts of interest to declare.

Interaction of Human Platelets with Enterohemorrhagic E. coli O111, Induces an Increase in the Procoagulant Activity, Thrombin Generation and Adhesion to Endothelial Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4989-4989
Author(s):  
Valeria Matus ◽  
José Guillermo Valenzuela ◽  
Claudia G Sáez ◽  
Patricia Hidalgo ◽  
Karla Pereira ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Clinical Manifestations ◽  
Lag Time ◽  
Human Platelets ◽  
Procoagulant Activity ◽  
Platelet Surface ◽  
E Coli ◽  
Time To Peak

Abstract Acute inflammation in response to severe bacterial infection, results in hemostatic abnormalities ranging from subclinical to sustained systemic clotting activation leading to massive thrombin and fibrin formation and microvascular thrombosis. Endothelial activation and dysfunction are critical determinants of the host response and provide an explanation for the different abnormalities involved in the pathophysiology of sepsis. Infection with pathogenic E. coli may present with a wide spectrum of clinical manifestations, from no symptoms or mild non-bloody diarrhea to severe cases, such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura, which is characterized by hemolytic anemia and low platelet counts. Although the understanding of the mechanisms that are involved in blood coagulation abnormalities in sepsis has gradually progressed, the role of platelets (Plts) on the procoagulant state during a severe infection remains to be addressed. Human platelets contain functional tissue factor (TF) (Panes et al. 2007) and TFPIa, but it is unknown if bacteria-platelet interaction affects platelet-TF procoagulant or platelet TFPI anticoagulant activities. Moreover, the effects of bacterial activation of platelets on thrombin generation (TG) in platelet rich plasma (PRP) or adhesion to endothelial cells have not been explored. Aims: We assessed the effect of platelet-E. coli interaction on platelet TF-dependent procoagulant activity (PCA), the changes induced by this interaction on platelet TFPI, in TG in PRP and in the adhesive capacity of platelets on cultured HUVEC. Plts activation by E. coli was demonstrated by a significant increase of p-selectin exposure on platelet surface compared to control Plts after 30 min of interaction with this microorganism harvested at exponential phase of growth and incubated in a ratio Plts/bacteria 1:10. Platelet TF-dependent PCA was assessed by FXa generation in washed Plts exposed to E. coli, with addition of exogenous FVIIa and FX, with no extra source of TF. Using the same ratio Plts/bacteria, we observed an increase in FXa after 30 min of incubation, compared with control platelets (p=0.0002, n=12). This enhancement in TF-PCA was concomitant with a decreased expression of TFPI in Plts surface after exposure of PRP to E. coli for 30 min. (p=0.0012, n=9). TG was measured in PRP, previously stimulated by E. coli for 30 min. We observed a shortening in the Lag time and time to peak and a higher thrombin peak in stimulated than in control PRP (p=0.0001, p=0.005 and p=0.0342, respectively, n=12). The reduction in lag time and time to peak was more pronounced than that obtained after eliciting platelet activation with Ristocetin. Preincubation of Plts with E. coli also increased the velocity index of TG compared to PRP alone (p=0,005; n=12). Static adhesion of Pts to endothelium was studied by stimulating fresh washed Plts with E. coli for 30min and then co-incubating them with HUVEC. After 20 min, an increased number of bacterial-activated Plts were adhered to HUVEC, compared with unstimulated Pts. Moreover, visible Plts aggregates were observed, which were positive for fibrin immunostaining, suggesting clot formation during the interaction of Plts with E. coli O111. Our findings show that Plts activated by bacteria results in an enhanced platelet procoagulant activity and adhesion to endothelium. By extension, these in vitro results suggest that platelets play an important role in the prothrombotic state associated with bacterial infections. This work was supported by FONDECYT-Chile 1130835 Disclosures No relevant conflicts of interest to declare.

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.

Platelet apoptosis and agonist-mediated activation in myelodysplastic syndromes

2013 ◽  
Vol 109 (05) ◽  
pp. 909-919 ◽  
Author(s):  
Víctor Jiménez-Yuste ◽  
Ihosvany Bello ◽  
Elena García Salgado ◽  
María Álvarez ◽  
Mónica Martín ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Myelodysplastic Syndromes ◽  
Thrombin Generation ◽  
Procoagulant Activity ◽  
Control Group ◽  
Bleeding Tendency ◽  
Western Blots ◽  
Platelet Apoptosis ◽  
Generation Capacity

SummaryPatients with myelodysplastic syndromes (MDS) have a defect in the differentiation of bone marrow multipotent progenitor cells. Thrombocytopenia in MDS patients may be due to premature megakaryocyte death, but platelet apoptotic mechanisms may also occur. This study aimed to study function and apoptotic state of platelets from MDS patients with different platelet count. Reticulated platelets, platelet activation, activated caspases and annexin-V binding were evaluated by flow cytometry. Pro-apoptotic Bax and Bak proteins were determined by western blots and plasma thrombopoietin by ELISA. Microparticle-associated procoagulant activity and thrombin generation capacity of plasma were determined by an activity kit and calibrated automated thrombography, respectively. High plasma thrombopoietin levels and low immature circulating platelet count showed a pattern of hypoplastic thrombocytopenia in MDS patients. Platelets from MDS patients showed reduced activation capacity and more apoptosis signs than controls. Patients with the lowest platelet count showed less platelet activation and the highest extent of platelet apoptosis. On this basis, patients with thrombocytopenia should suffer more haemorrhagic episodes than is actually observed. Consequently, we tested whether there were some compensatory mechanisms to counteract their expected bleeding tendency. Microparticle-associated procoagulant activity was enhanced in MDS patients with thrombocytopenia, whereas their plasma thrombin generation capacity was similar to control group. This research shows a hypoplastic thrombocytopenia that platelets from MDS patients possess an impaired ability to be stimulated and more apoptosis markers than those from healthy controls, indicating that MDS is a stem cell disorder, and then, both number and function of progeny cells, might be affected.

How I Manage cyclic thrombocytopenia

Blood ◽  
2020 ◽  
Author(s):  
Paul A Kyrle ◽  
Sabine Eichinger
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenia ◽  
Hematological Malignancies ◽  
Severe Bleeding ◽  
Typical Pattern ◽  
Periodic Fluctuation ◽  
Thrombopoietin Receptor ◽  
Primary Immune Thrombocytopenia ◽  
Platelet Count Monitoring ◽  
Mucocutaneous Bleeding

Cyclic thrombocytopenia (CTP) is a rare disease, which is characterized by periodic fluctuation of the platelet count. The pathogenesis of CTP is unknown and most likely heterogeneous. Patients with CTP are almost always misdiagnosed as having primary immune thrombocytopenia (ITP). The interval between ITP and CTP diagnosis can be many years. CTP patients often receive ITP-specific therapies including corticosteroids, thrombopoietin receptor agonists, rituximab and splenectomy which are followed by a transient increase in platelet count that is wrongly attributed to treatment effect with inevitable "relapse". CTP can be diagnosed by frequent platelet count monitoring which reveals a typical pattern of periodic platelet cycling. An early diagnosis of CTP will prevent these patients from being exposed to possibly harmful therapies. The bleeding phenotype is usually mild and consists of mucocutaneous bleeding at the time when the platelet count is at its nadir. Severe bleeding from other sites can occur but is rare. Some patients respond to cyclosporine A or to danazol, but most patients do not respond to any therapy. CTP can be associated with hematological malignancies or disorders of the thyroid gland. Nevertheless, spontaneous remissions can occur, even after many years.

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