Red-Cell Microparticles Released From Stored Packed Cells: Possible Contributing Factor to Adverse Responses to Transfusion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 342-342 ◽  
Author(s):  
Wenche Jy ◽  
Carlos Bidot ◽  
Max E Johansen ◽  
Lawrence Horstman ◽  
Sherry Shariatmadar ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Blood Bank ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Blood Type ◽  
Storage Lesion ◽  
Rate Of Increase ◽  
Proinflammatory Activity

Abstract Abstract 342 Background. Packed red cells (PC) stored in blood bank undergo a series of changes, so-called “storage lesion”, which increase with time. In view of some but not all recent studies, it is widely believed that transfusion with younger blood carries less risk of adverse reactions than older blood. However, there is no agreement on the “safe” age of blood, nor is it clearly understood why older blood may carry increased risks. The purpose of this study was to identify microparticle-related factors in stored PC at different time intervals that might pose risk of adverse effects. We investigated profiles of cell-derived microparticles (MP), particularly RBC-derived MP (RMP), in stored PC and assessed their procoagulant and inflammatory property. Methods. Twelve bags of fresh packed red cells (PC) of known blood types (A+, B+, AB+, O+) were obtained from blood bank (2-4 days since drawing). All were non-leuko depleted and were stored at 4°C. Time of receipt was considered day 0. At intervals of 0, 10, 20, and 30 days, 40 mL was withdrawn and centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for (1) quantity of different species of cell-derived MP by flow cyteometry comprising (a) RMP defined by CD235b; (b) LMP by CD45; (c) PMP by CD41; (d) EMP by CD144; (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV), (2) procoagulant activity by MP-mediated thrombin generation assay (TGA); (3) MP-mediated proinflammatory activity by CD 11b expression in neutrophils following incubation with RMP. Results. (1) MP Profiles. The time-course of generation of the MP subtypes varied considerably. For RMP, there was little increase before day 10, but then rose rapidly with time, to 180% at 20 days, and to 450% at 30 days. Small amounts of MP derived from leukocytes (LMP), platelets (PMP), and endothelia (EMP) were present in all bags at day 0, generally <10% by number compared to RMP. For LMP, there was no significant change in the first 20 days but was increased significantly at day 30, to 160% of day 0. For PMP, counts rose steadily from day 0 and peaked to 220% of baseline at day 20. For EMP, counts were very low (<1% of RMP) and no change was observed over 30 days. For total MP, defined by Ulex counts and total protein concentrations, the time course was similar to RMP. Results with AnV+ MP showed significant increase from day 10 to day 20. We found no influence of blood type on MP generation. (2) Procoagulant Activity. There was little change in MP-mediated thrombin generation in the first 10 days, but it rose significantly from day 10 to day 20, correlating well with counts of AnV+ MP or RMP. (3) Proinflammatory Activity. Leukocyte CD11b expression induced by MP from the PC bags showed a nearly linear rate of increase from day 0 to day 30, correlating closely with PMP. (4) Exceptions. In 2 of the 12 PC bags, we observed exceptionally high levels of both RMP and PMP (4-8 fold higher than average) from day 0 to day 30. CBC assay showed that all bags contained similar counts of RBC and WBC, with exception of high concentrations of platelets (>200,000/μL) in the two exceptional bags vs. the others (20,000 – 45,000/μL). In the two exceptional bags, coagulant activities and inflammatory potential were also highly elevated compared to the values in the other bags from day 0 to day 30. Conclusions. (i) RMP are the predominant MP species in stored RBC, increasing slowly from day 0 to 10 and thereafter rising exponentially to day 30. (ii) PMP were also present in significant amounts. The time course of RMP and PMP release correlated well with procoagulant and proinflammatory indicators in stored RBC. (iii) Two of the 12 bags (17%) exhibited exceptionally high platelet content and RMP, PMP. The significance of this new finding remains to be clarified. These preliminary results indicate that procoagulant, proinflammatory MP levels increased significantly after 10 days of storage, and that contaminating platelets exacerbate RMP generation. The increase in MP in stored PC constitutes one aspect of the storage lesion and may pose prothrombotic and/or proinflammatory risks in blood transfusions. (Supported by NIH Grant 5R01HL098031-02) Disclosures: No relevant conflicts of interest to declare.

Red Cells Release Microparticles Heterogeneous in Phenotypes, Procoagulant Activities, and Size under Different Conditions.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1726-1726
Author(s):  
Wenche Jy ◽  
Jaehoon Bang ◽  
Joaquin J. Jimenez ◽  
Lawrence L. Horstman ◽  
Loreta Bidot ◽  
...  
Keyword(s):  
Shear Stress ◽  
Thrombin Generation ◽  
Calcium Ionophore ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Ionophore A23187 ◽  
Flip Flop ◽  
Increased Risk ◽  
Thrombotic Complications ◽  
Inside Out

Abstract BACKGROUND. Hemolytic anemias (HA) such as autoimmune hemolytic anemia (AIHA), thalassemia, and sickle cell disease are associated with increased risk for thrombosis. Although exposure of procoagulant phospholipids on the RBC membrane in HA has been implicated, the pathogenesis remains to be elucidated. We recently showed that microparticles released from red cells (RMP) are procoagulant and significantly elevated in HA, suggesting a link of RMP to thrombotic complications. In this study, we report the release of RMP, heterogeneous in size, phenotypes and procoagulant activity by different methods. METHODS. RMP were prepared from washed RBC (2×109/mL) by three methods, exposure to 10 μM calcium ionophore A23187 (CaIo) for 60 min, 20 μM lysophosphatidic acid (LPA) for 60 min, or shear stress induced by a rotating Teflon pestle for 5 min at 160 rpm in a tissue grinder. After removing residual whole cells, the supernatant was centrifuged at 15,000×g for 10 min (large RMP) and the supernatant was further centrifuged at 150,000×g for 30 min (small RMP). The resulting RMP were labeled for flow cytometry using PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). Thrombin generation of RMP was measured by the calibrated automated thrombogram (CAT) system using fluorescent thrombin substrate on a fluorescence plate reader. RESULTS. The two-step centrifugation revealed two distinct populations, the large RMP expressing both GlyP and Ulex binding while the smaller RMP expressed only Ulex binding. Electron micrography showed diameters for the large RMP of 200 - 800 nM while the small RMP were 40 - 80 nm. Shear stress produced the greatest number of large GlyP + RMP (5.2 ± 1.3 × 106/μL), followed by CaIo (3.8 ± 0.7 × 106/μL), and LPA (2.5 ± 0.6 × 106/μL). However, CaIo produced the greatest number of small GlyP −/Ulex + RMP (4.6 ± 0.9 × 106/μL), followed by LPA (1.4 ±0.3 × 106/μL), and very few by shear tress (0.3 ±0.1 × 106/μL). The methods also gave different AnV binding: CaIo yielded &gt;90% of RMP (large and small) that were positive, whereas RMP induced by LPA or shear stress gave only 45% and 18% positive, respectively. No TF + RMP were detected in any procedure. The RMP also differed in thrombin generation. Adjusting concentrations to equal numbers of Ulex + MP, CaIo RMP displayed the strongest activity (375 ± 62 nM) followed by LPA-induced (227 ± 58 nM) and shear-induced (136 ±33 nM). Thrombin generation correlated well with degree of AnV binding. CONCLUSIONS. We demonstrate that RBC release different species of RMP, heterogeneous in size, phenotypes and procoagulant activity. Both CaIo and LPA induced two distinct species of RMP, the larger expressing GlyP and Ulex binding while the smaller phenotype was negative for GlyP, possibly indicating that the small RMP are inside-out vesicles. In contrast, shear stress produced mainly large rightside-out vesicles with both GlyP expression and Ulex binding, and very low AnV binding. Thus, calcium influx appears necessary to release small inside-out RMP, and to induce the membrane flip-flop bringing negatively charged AnV binding sites to the plasma membrane. We postulated that highly procoagulant RMP that bind AnV may contribute to the thrombotic complications of HA and other RBC disorders associated with thrombosis.

Single Nucleotide Variations In Spectrin-1β Accentuate The Red Blood Cell Storage Lesion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3422-3422
Author(s):  
Melinda M Dean ◽  
Katrina Kildey ◽  
Thu V Tran ◽  
Kelly Rooks ◽  
Shoma Baidya ◽  
...  
Keyword(s):  
Time Course ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Osmotic Fragility ◽  
Blood Component ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Storage Lesion ◽  
Background Strain ◽  
The Impact

Abstract Introduction During routine storage packed red blood cells (PRBC) undergo biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. Donor-to-donor variability in the severity of the storage lesion has been reported. The extent to which donor-associated differences in blood component storage affect blood product quality and post-transfusion outcome remains unknown. Murine models with single nucleotide variants (SNV) in gene encoding spectrin-1β were used to investigate the impact of mutations on the RBC storage lesion. Methods Two murine lineages with N-ethyl-N-nitrosourea (ENU) generated single SNV in Spnb1, encoding spectrin-1β (Table 1), were selected from the Australian Phenomics Facility library (http://databases.apf.edu.au/mutations). Using genetic selection, homozygous (HOM), heterozygous (HET) and unaffected (WT) mice from each strain were generated (C57BL/6 background strain). Murine blood was leucoreduced, prepared in SAGM (0.4 HCT) and stored at 4°C for time course assessment of RBC characteristics. At day (D), D2, D7, D14 and D21 of storage, RBC integrity and evidence of storage-related changes were investigated using RBC osmotic fragility and flow cytometric analysis of CD44, CD47, TER119 and phosphatidylserine (PS). Data were generated from analysis of blood from Spnb1 (pedigree spectrin-1β a) homozygous (HOM, n=3), heterozygous (HET, n=3) and unaffected (WT, n=2 ); Spnb1 (pedigree spectrin-1β b) HOM (n=4), HET( n=4); C57BL/6 (n=4). The Mann-Whitney Test and ANOVA were utilised for statistical analyses (95% CI). Results At D2 of storage SNV in Spnb1 did not alter RBC characteristics, with all mice studied demonstrating a similar resistance to osmotic lysis and levels of CD44, CD47, TER119 and PS. By D7 of storage, clear pedigree-related differences in RBC characteristics were evident. At D7, RBC from spectrin-1β(a) HOM mice had significantly increased osmotic fragility and exposure of PS as well as significantly reduced CD44 and TER119 expression compared to unaffected siblings and background strain. Of note, these changes were not evident in the spectrin-1β(b) HOM mice at D7. For both strains at D7, heterozygous SNV did not exhibit altered storage parameters. By D14 both HOM and HET spectrin-1β(a) mice demonstrated a phenotype consistent with an exacerbated RBC storage lesion, characterised by significantly increased osmotic fragility and exposure of PS, and reduced CD44 and CD47 compared to background strain. At D14 there was also evidence of exacerbation of the storage lesion in stored RBC from HOM spectrin-1β(b) mice (significantly increased PS), though this was not to the extent observed in the spectrin-1β(a) mice. By D21 all murine RBC were substantially degraded under these storage conditions. Conclusions SNV in Spnb1,encoding RBC structural protein spectrin-1β, resulted in both early onset and exacerbation of the RBC storage lesion. Further, the degree of storage lesion and the point at which RBC degradation was observed was not only dependent on the homozygous or heterozygous status, but the mutation itself. These data demonstrate that minor genetic variation in genes encoding important RBC proteins contribute to donor related differences in PRBC storage. Disclosures: No relevant conflicts of interest to declare.

Procoagulant Status In Patients With Immune Thrombocytopenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3528-3528 ◽  
Author(s):  
Ihosvany Fernández Bello ◽  
Mayte Álvarez Román ◽  
Elena G. Arias Salgado ◽  
Monica Martin Salces ◽  
Miguel Canales ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Red Cells ◽  
Procoagulant Activity ◽  
Platelet Counts ◽  
Significant Difference ◽  
The Mean ◽  
Spearman Test

Abstract Introduction Immune thrombocytopaenia (ITP) is an acquired immune-mediated disorder characterized by mild to severe thrombocytopaenia caused by autoantibodies against platelet proteins. Bleeding risk in patients with ITP is increased with platelet counts less than 20 or 30 x 109/L. However, patients with ITP often have few bleeding symptoms despite very low platelet counts suggesting the existence of compensatory mechanisms. Moreover, an increased risk for thrombosis in patients with ITP has been described (Nørgaard M, 2012). It has been recently reported that increased production of platelet- and red cells-derived microparticles (MP) might be one of the causes of increased thrombotic risk in ITP patients (Sewify, 2013). Objective The aim of this study was to evaluate the microparticle-associated and plasma procoagulant activities in ITP patients with thrombocytopaenia. Methods Sixty-eight patients with chronic ITP and platelet count less than 50 x 109/L and twenty-two healthy controls were included. Platelet counts were determined with a Coulter Ac. T Diff cell counter (Beckman Coulter, Madrid, Spain). Citrated blood was centrifuged at 1,500 g for 15 min at 23°C. Platelet-poor plasma obtained was additionally centrifuged twice at 23°C (15 min at 1,500 g, and 2 min at 13,000 g) and aliquots were stored at -70ºC until analysis. Phosphatidylserine-MP (Ph-MP) and tissue factor-MP (TF-MP) dependent procoagulant activities were determined with the ZYMUPHEN kits (Hyphen BioMed, Neuville sur Oise, France) following the manufacturer’s instructions. Plasma thrombin generation was measured using the Calibrated Automated Thrombogram (CAT) test as described by Hemker et al (2000) at a final concentration of 1 pM tissue factor and 4 μM phospholipids (PPP-Reagent LOW, Thrombinoscope BV, Maastricht, The Netherlands). We evaluated the endogenous thrombin potential (ETP, the total amount of thrombin generated over time); the lag time (the time to the beginning of the explosive burst of thrombin generation); the peak height of the curve (the maximum thrombin concentration produced); and the time to the peak. To test resistance to protein C, CAT experiments were performed without and with the addition of thrombomodulin (TM) (PPP and PPP with thrombomodulin reagents, Thrombinoscope BV, Maastricht, The Netherlands). Results were expressed as the ratio [(ETP with TM)/(ETP without ETP)]x100. Results were expressed as mean±SD. Comparisons of quantitative variables were made with Mann-Whitney test and correlations with Spearman test. Values of p≤0.05 were considered statistically significant. Results Ph-MP associated procoagulant capacity in ITP patients was higher than in controls (p<0.05) whereas MP-TF associated procoagulant activity was practically negligible in both groups. Plasma procoagulant activity was higher in ITP patients than in controls (ETP: 1604±616 nM x min in ITP patients and 1302±416, p=0.012 in controls; Peak: 328±123 nM in ITP patients and 203±74 nM in controls, p<0.001). We tested whether the higher procoagulant activity of plasma from ITP patients was due to a resistance to protein C. We observed that the mean Ratio value in ITP patients was slightly higher than the mean Ratio of controls (60±18 and 50±13 respectively, p=0.034). Despite this significant difference in the Ratio, no correlation was found between this value and the CAT parameters. Conclusion ITP patients with thrombocytopaenia had a higher Ph-MP associated and plasma procoagulant activity than controls. The fact that the increased MP-procoagulant activity was not accompanied by a higher TF-MP associated procoagulant activity brings further support to the previous observation that MPs in ITP patients are from platelets and red cells, as both cells express very low levels of TF (Sewify, 2013). Regarding the increased plasma procoagulant capacity observed in ITP patients, our results suggest that resistance to protein C does not seem to be the main mechanism involved. References • Nørgaard M. Thromb Res. 2012;130 Suppl 1:S74-75. • Sewify EM, et al. Thromb Res. 2013;131:e59-63. Hemker HC, et al. Thromb Haemost 2000;83:589-9. Disclosures: No relevant conflicts of interest to declare.

Persistant Inhibition of Thrombin Generation After Intravenous Administration of Enoxaparin in Primates.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2095-2095
Author(s):  
Evangelos Litinas ◽  
Angel Gray ◽  
Nasir Sadeghi ◽  
Josephine Cunanan ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Clinical Effects ◽  
Plasma Samples ◽  
Thrombin Inhibition ◽  
Biologic Effects ◽  
Antithrombotic Effects ◽  
Sustained Inhibition ◽  
Inhibition Of Thrombin

Abstract Abstract 2095 Poster Board II-72 The biologic half life (T12) of low molecular weight heparin (LMWH) is usually measured in terms of the circulating anti-Xa levels. Enoxaparin represents an unique LMWH whose biologic T12 is relatively longer than most LMWHs. Moreover, it is known that the antithrombotic effects of this agent last longer in comparison to the measurable circulating anti-Xa levels. Therefore besides the anti-Xa activity, additional non-measurable biologic effects are contributory to the clinical effects of this agent. Plasma based thrombin generation assays have recently become available to assess the effects of LMWHs such as enoxaparin. In these assays blood plasma samples are activated using different activators and the generated thrombin inhibition is measured. To measure the time course of thrombin generation inhibitory activity after an IV bolus dose of 0.5 mg/kg of enoxaparin into groups of primates (n=6-8), a commercially available thrombin generation method was employed (Technoclone, Vienna, Austria/DiaPharma, West Chester,OH). Blood samples were drawn from each of the primates injected at varying time points for up to 28 hours. A thromboplastin/phospholipids based reagent was used to generate thrombin and the results were recorded in terms of nm of thrombin formed. The baseline values ranged from 500-900 nm (710±60 nm), although a complete inhibition of thrombin generation was noted at 1 hour (24±8 nm), a slow and gradual reduction in the thrombin generation inhibition was noted with a T12 of 9 hours. Even at 28 hours after the administration of enoxaparin, sustained inhibition of thrombin generation was noted (30-50%). Interestingly, the circulating anti-Xa and anti-IIa activity gradually diminished to an almost non-detectable level at 6 hours. These studies suggest that enoxaparin produces antithrombotic actions by multiple mechanisms. Furthermore thrombin generation methods in plasma samples may provide a more sensitive assay for the monitoring of the effect of LMWH. Disclosures: No relevant conflicts of interest to declare.

A Novel Mechanism of Action of Rivaroxaban: Inhibition of Monocyte and Macrophage Procoagulant Activity and Consequence On Inflammatory Process.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3124-3124
Author(s):  
Marc Laurent ◽  
Remi Varin ◽  
Ulrich Joimel ◽  
Hong Li ◽  
He Lu ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Moderate Increase ◽  
Amidolytic Activity ◽  
Monocytes And Macrophages ◽  
Activated Monocytes ◽  
Prothrombinase Activity

Abstract Abstract 3124 Poster Board III-61 Aim of the study Tissue factor is normally absent from monocytes in circulating blood. It can be induced by inflammatory mediators leading to the formation of monocyte-associated prothrombinase activity, which participates to thrombin generation. This activity appears to be important in both thrombosis (venous and arterial) and in chronic inflammation by inducing the release of inflammatory cytokines. This could be attributed to the activation of PAR-1 and PAR-2 (protease-activated receptors) by factor Xa or by thrombin. In this study, we compared the action of Rivaroxaban and Fondaparinux, two specific factor Xa inhibitors, on the activation of coagulation and on the secretion of inflammatory cytokines in both activated monocytes and activated human monocyte/macrophage cell line THP1. Methods 1-Monocytes were isolated from healthy volunteers and THP-1 cells were used as macrophages. 2- Activation of cells was performed by adding 1 μg/ml LPS for 2 hours at 37°C, in the presence of defibrinated human plasma which provides plasma coagulation factors, in the absence (control) or presence of Fondaparinux (500, 1000 and 1500 ng/ml) or Rivaroxaban (150, 250, 350 ng/ml, final concentrations). After incubation, the cells were isolated and their supernatants collected. 3- The procoagulant activity of cells was tested by measuring their effect on the clotting time of normal plasma in presence of calcium. 4- The release of cytokines was tested by antibody-cytokines array in the supernatants (RayBio®). 5-To test the neutralization of factor Xa bound to monocytes, activated monocytes were incubated with human purified factor Xa (100 ng/ml) for 10 min at room temperature. After isolation of the cells, 250 ng/ml Rivaroxaban or the appropriate solvent was added for 1, 10 and 30 min. The Xa activity associated to monocytes was measured by its amidolytic activity. Results 1- The procoagulant activity of monocytes and macrophages was reduced by Rivaroxaban and not by Fondaparinux. Results of prothrombinase activity in the presence of Rivaroxaban at 150, 250, 350 ng/ml, expressed as percentage of the control value was: 30 ± 3, 16 ± 4 and 12 ± 2 % for monocytes and: 43 ± 2, 24 ±3, 15 ± 1 % for THP-1 cells. In contrast, Fondaparinux did not modify the prothrombinase activity of monocytes (105 ± 12%). It was also shown that in the conditions used, Rivaroxaban (250 ng/ml) inhibited completely the amidolytic activity of factor Xa bound to activated monocytes. 2- Both Rivaroxaban and Fondaparinux modify the profile of chemokines secretion by activated monocytes and THP-1. In monocytes and THP-1 cells, LPS induced an important increase in Il-8 and angiogenin and a moderate increase in MIP-1d (Macrophage Inflammatory Protein-1) and RANTES, a member of the Il-8 superfamily. Both Rivaroxaban and Fondaparinux decreased the secretion of these chemokines to the basal level of secretion by non activated monocytes or THP-1. The secretion of leptin was only induced by LPS- treated THP-1 and was strongly decreased by both FXa inhibitors. In contrast, secretion of EGF (epithelial growth factor) was only induced by activated monocytes and strongly decreased by Rivaroxaban and moderately by Fondaparinux. Discussion and conclusion The results show that 1- Rivaroxaban induced a concentration-dependent inhibition of the procoagulant activity of activated monocytes and macrophages, whereas fondaparinux was devoid of this effect. This difference is attributed to a better access of Rivaroxaban to FXa bound to monocytes, as compared to the Fondaparinux-antithrombin complex. This inhibitory effect of Rivaroxaban could contribute to its antithrombotic activity. 2- Rivaroxaban inhibited the secretion of inflammatory chemokines by activated monocytes and THP-1. This decreased secretion was also observed with Fondaparinux, suggesting that it could be due to the decrease in thrombin generation in plasma, affecting the PAR-1 cell signaling system. As it was reported that that elevated IL-8 is associated with recurrent venous thrombosis and that these inflammatory cytokines are involved in plaque progression and rupture by recruitment of subpopulations of leukocytes and by potent angiogenic activity, this decrease in cytokines could also contribute to the antithrombotic efficacy of Rivaroxaban. This study received a support from Bayer Schering Pharma, France. Equal contribution of Marc Laurent and Rémi Varin Disclosures No relevant conflicts of interest to declare.

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.

Donor Variation In Biological Mediators During Storage Of Packed Red Blood Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3655-3655 ◽  
Author(s):  
Melinda M Dean ◽  
Luke D Samson ◽  
Kelly Rooks ◽  
Jesse Fryk ◽  
Shoma Baidya ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Immune Modulation ◽  
Time Course ◽  
Conflicts Of Interest ◽  
Biological Response ◽  
Storage Lesion ◽  
Packed Red Blood Cells ◽  
Donor Variation ◽  
Time Point

Abstract Introduction During routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. Methods Cytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). Results Of the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P<0.001, ANOVA). Of particular interest, the concentrations of these nine biological mediators varied greatly between the individual PRBC units. ICAM-1, VCAM-1 and IL-1α concentrations each varied 10 fold between units (range 1000 – 10 000 pg/mL for each), sCD14 varied 5 fold (range 20 000 - 100 000 pg/mL), sCD62L varied 4.4 fold (range 9000 – 40 000 pg/mL), and sCD62P varied 6.5 fold (range 200 -1300 pg/ml). In addition, it was apparent that a sub population (3/8) of the units assessed consistently had the highest levels of ICAM-1, sCD106 (VCAM-1), sCD14, sCD62L, IL-1α, sCD62P and angiogenin. For sCD62P, in particular, this “high” sub population had significantly different levels of sCD62P at each time point compared to the other five units (P<0.05 at each time point). The remaining BRMs studied were at the limits of detection (<20 pg/mL) for every unit at each time point, and no storage related changes were evident. Conclusions There was minimal change in the BRMs studied relevant to storage duration of the PRBC units. The most notable differences in the levels of biological mediators present in PRBC SN were due to donor-to-donor variation. These data suggest high levels of BRMs and potential immune modulation in transfusion recipients may be the result of donor-associated differences rather than storage-associated differences in blood components. Disclosures: No relevant conflicts of interest to declare.

Comparison of the Procoagulant Activity of Freeze-Dried and Fresh Platelets with Phospholipid in Three Phospholipid Dependent Assays.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3990-3990
Author(s):  
Elaine Gray ◽  
William M. Pickering ◽  
Alison H. Goodall ◽  
Trevor W. Barrowcliffe
Keyword(s):  
Thrombin Generation ◽  
Time Course ◽  
Activity Patterns ◽  
Bovine Brain ◽  
Procoagulant Activity ◽  
In Vitro Tests ◽  
Healthy Donors ◽  
Freeze Dried ◽  
And Function

Abstract Background and Objective: Platelet activation and coagulation together determine the haemostatic activity of plasma. When activated, platelets expose phosphatidylserine (PS) at their outer surface, on which the tenase and prothrombinase complexes assemble, forming FXa and thrombin, respectively. In in-vitro tests phospholipid vesicles are normally used as a platelet substitute. However it is not clear if PL vesicles behave in the same way as platelets. The objective of the study was to compare and contrast the procoagulant activity (PCA) of lyophilized platelets, a bovine brain phospholipid standard (NIBSC 91/542) against fresh platelets using three phospholipid (PL) dependent assays. Inhibition of this PCA by Annexin A5 (AA5) and an anti-PS antibody (3G4) was also studied. Methods and Materials: Lyophilised fixed platelets were obtained from Bio/Data corporation. Fresh platelets were collected into ACD-A from healthy donors and then gel-filtered through Sepharose 2B. Fresh platelets were activated by A23187 (10μM) before use in the assay systems. Platelet counts over a range of 10–200 x 109/L were used in the assays. FXa and prothrombinase generation were measured in purified chromogenic systems, and calibrated against FXa and IIa standards. The time course of thrombin generated in plasma activated by FIXa was monitored, and peak thrombin levels and the total amount of free thrombin measured over time (ETP). AA5 and 3G4 at concentrations of 3.3–33 μM and 50–400 μg/mL respectively were used to inhibit the assembly and function of macromolecular coagulant enzyme complexes. Results: Fresh platelets were 2.3 and 2.8-fold more potent than lyophilized platelets and PL standard respectively, in supporting FXa generation. When prothrombinase activity was measured, fresh platelets were 3.4 and 38-fold more potent than the lyophilized platelets and PL standard, respectively. Lyophilised platelets were 11-fold more potent than the PL standard. When thrombin generation was investigated fresh platelets were 3.1 (peak thrombin) and 3.7-fold (ETP) more potent than lyophilized platelets and 2.2 (peak thrombin) and 7-fold (ETP) more active than the PL standard. Lyophilised platelets were 0.7 (peak thrombin) and 1.9-fold more active than the PL standard. The tenase and prothrombinase PCA supported by the PL standard and fresh platelets were more susceptible to AA5 (8.3μM) inhibition (>70%) than lyophilized platelets (45 – 59%) though this activity was totally inhibited at 33μM. In the same tests 3G4 (400μg/mL) completely inhibited the PL standard but was only able to inhibit fresh and lyophilized platelets by 40–80%. Thrombin generation supported by the PL standard was completely inhibited by AA5 (16.5μM). Lyophilised platelets were more susceptible to the action of AA5 than fresh platelets. However 3G4 (400μg/mL) was unable to completely inhibit thrombin generation activity supported by PL standard or platelet preparations. 3G4 for all preparations delayed the time taken for peak thrombin levels to be reached. Conclusions: Lyophilized platelets showed similar activity patterns to the PL standard in supporting tenase and thrombin formation/generation, though they were more active than the PL standard. Fresh-platelets were significantly better able to support these complexes, probably because of the presence of specific receptors for FVa and FXa and the maintenance of membrane fluidity.

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.

scholarly journals Preferential Stimulation of Contact Pathway Factors By Red Cell-Derived Microparticles: New Perspective on Hemostasis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4234-4234
Author(s):  
Max E Johansen ◽  
Wenche Jy ◽  
Lawrence L Horstman ◽  
Yeon-Soong Ahn
Keyword(s):  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Procoagulant Activity ◽  
Red Cell ◽  
High Concentration ◽  
Extrinsic Pathway ◽  
Excessive Bleeding ◽  
Low Concentration ◽  
Contact Pathway ◽  
Stimulation Of

Abstract BACKGROUND / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly. METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP. RESULTS. (1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p<0.01 to p<0.05, at depletions as low as 5% normal. For example, FVIII at 15% of normal level responded to RMP by 50% reduction in R time. In contrast, RMP had little or no augmenting effect on plasmas depleted of F’s II, V, X. This suggests that RMP promote the contact pathway preferentially. (2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation. (3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p<0.05). This demonstrates that RMP can potentiate thrombin generation via the TF pathway, although not as robustly as by contact pathway. CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration. Disclosures No relevant conflicts of interest to declare.

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