Anja Leyte

Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia

Essential thrombocythemia is a myeloproliferative neoplasm characterized by an increased risk of both arterial and venous thrombosis. The findings of this study show that patients with this disorder have elevated levels of platelet-and endothelium-derived microparticles, which may support thrombin generation and play a role in the pathophysiology of thromboembolic complications. Background Most cell types, including blood - and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia. Design and Methods In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay. Results Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500×106 events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001). Conclusions Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.

Pravastatin reduces fibrinogen receptor gpIIIa on platelet‐derived microparticles in patients with type 2 diabetes

*Laboratory for Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam; Department of InternalMedicine, Slotervaart Hospital, Amsterdam; Laboratory of Haematology and Clinical Chemistry, Onze Lieve Vrouwe Gasthuis, Amsterdam; and§Department of Internal Medicine and Cardiovascular Research Institute Maastricht, Academic Hospital and University of Maastricht, Maastricht,The NetherlandsTo cite this article: Sommeijer DW, Joop K, Leyte A, Reitsma PH, ten Cate H. Pravastatin reduces fibrinogen receptor gpIIIa on platelet-derivedmicroparticles in patients with type 2 diabetes. J Thromb Haemost 2005; 3: 1168–71.See also Davi` G, Ferroni P. Microparticles in type 2 diabetes mellitus. This issue, pp 1166–7.

Measuring Rivaroxaban in a clinical laboratory setting, using common coagulation assays, Xa inhibition and thrombin generation.

Abstract Background: Rivaroxaban, a direct Xa inhibitor, is one of the new oral antithrombotic agents for which laboratory monitoring is thought to be unnecessary in most cases due to predictable pharmacokinetics. Circumstances are conceivable, however, in which reliable laboratory testing of Rivaroxaban is desirable. The aim of the present in vitro study was to investigate and compare the analytical and practical use of Rivaroxaban monitoring with routine screening assays, thrombin generation and anti-Xa activity, in a clinical laboratory setting. Methods: Rivaroxaban was added to nine normal donor plasmas and to a normal pooled plasma in concentrations up to 1000 μg/L. Prothrombin time (PT), activated partial thromboplastin time (APTT), endogenous thrombin potential (ETP) and anti-Xa activity were measured in all donor samples. Responsiveness to Rivaroxaban and imprecision of Rivaroxaban recovery were assessed. Results: Low intra-, but high inter-individual imprecision was found for PT displaying a linear dose-response relationship. Imprecision was much lower when directly measuring anti-Xa activity. Responsiveness of ETP lag-time was high, but of total thrombin generation was low, illustrating that the main effect of Rivaroxaban Xa inhibition lies in delaying thrombin formation rather than in preventing it. Conclusions: Despite a high inter-individual imprecision of the PT, this relatively fast and cost-friendly assay is sensitive to Rivaroxaban and integrates its effects on the global coagulant state of patients. Anti-Xa activity assays can be run to assess the actual Rivaroxaban concentration and in the future ETP could serve as a fine-tuned hemostatic balance indicator for patients using Rivaroxaban.

Circulating white blood cells and platelets amplify oxidative stress in heart failure.

Background Mitochondria of circulating white blood cells (WBC) and platelets sense oxidative stress during capillary passage and react by producing reactive oxygen species (ROS). Although evidence indicates that congestive heart failure (CHF) is associated with oxidative stress, the role of WBC and platelets as mediators in CHF has not been investigated.Methods Patients with CHF (n = 15) and healthy volunteers (n = 9) were enrolled between 2006 and 2007 into this observational study. Arterial and venous blood samples from participants were incubated with probes to detect cytosolic and mitochondrial ROS. Fluorescence-activated cell sorting was used to measure the degree of fluorescence in WBC and platelets.Results Patients with CHF had a higher proportion of ROS-positive arterial WBC and platelets than did controls (67% ± 47% versus 16% ± 9%; P <0.005), as well as venous WBC and platelets (77% ± 43% versus 38% ± 13%; P <0.01). In the control group, the proportion of cytosolic ROS-positive arterial WBC and platelets was lower than that for ROS-positive venous WBC and platelets (16% ± 9% versus 38% ± 13%; P <0.005). CHF patients had a higher proportion of mitochondrial ROS-positive arterial and venous WBC and platelets than did controls.Conclusion In CHF, the proportion of WBC and platelets that are ROS-positive is raised, possibly because cytosolic ROS-positive WBC and platelets are normally cleared in the lungs; this function is deficient in CHF while mitochondrial ROS production is increased. The raised numbers of circulating ROS-positive WBC and platelets amplify oxidative stress in CHF.

Global coagulation tests: their applicability for measuring direct factor Xa- and thrombin inhibition and reversal of anticoagulation by prothrombin complex concentrate

Abstract Background: Specific mass spectrometry and direct activated factor X (Xa)- and thrombin inhibition assays do not allow determination of the reversal of anticoagulant effects of non-vitamin K direct oral anticoagulants (NOACs) by prothrombin complex concentrate (PCC). The objective of this study was the evaluation of the applicability of a variety of commercially available global coagulation assays in analyzing the reversal of NOAC anticoagulation by PCC. Methods: Plasma and whole blood were spiked with apixaban or dabigatran and PCC was added to these samples. Prothrombin time (PT), modified PT (mPT), activated partial prothrombin time (APTT), thrombography (CAT method) and thromboelastography (ROTEM, TEG) were performed. Results: Assays triggered by contact activation (APTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor (TF) showed NOAC type and NOAC concentration dependent anticoagulation reversal effects of PCC ranging from partial normalization to overcorrection of the following parameters: clotting or reaction time (PT, mPT TEG-TF, EXTEM, FIBTEM); angle in thromboelastography (TEG-TF); thrombin generation (CAT) lag time, endogenous thrombin potential (ETP) and peak thrombin. Extent of reversal was assay reagent dependent. ETP (5 pM TF) was the only parameter showing complete reversal of anticoagulation by PCC for all NOACs ranging from 200 to 800 μg/L. Conclusions: ETP fits with the concept that reversal assessment of NOAC anticoagulation by PCC should be based on measurements on the clotting potential or thrombin generating potential of the plasma or whole blood patient sample. Low sensitivity of ETP for NOACs and its correlation with bleeding are issues that remain to be resolved.

Procoagulant myeloblast-derived microparticles in AML patients: changes in numbers and thrombin generation potential during chemotherapy

M. C . V AN AALDEREN ,* 1 M. C . T RAPP ENB URG,* 1 M. VA N SCHILF GA ARDE , P . J . MOLENAAR , H. TEN C ATE , W. E . TERPSTRA* and A . LEY TE *Department of Internal Medicine, and Department of Clinical Chemistry, Onze Lieve Vrouwe Gasthuis, Amsterdam; and Department of Internal Medicine and Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands

Increased PAI-1 plasma levels and risk of death from dengue: no association with the 4G/5G promoter polymorphism

BackgroundDengue virus infected patients have high plasminogen activator inhibitor type I (PAI-1) plasma concentrations. Whether the insertion/deletion (4G/5G) polymorphism in the promotor region of the PAI-1 gene is associated with increased PAI-1 plasma concentrations and with death from dengue is unknown. We, therefore, investigated the relationship between the 4G/5G polymorphism and PAI-1 plasma concentrations in dengue patients and risk of death from dengue.MethodsA total of 194 patients admitted to the Dr. Kariadi Hospital in Semarang, Indonesia, with clinical suspected severe dengue virus infection were enrolled. Blood samples were obtained on day of admission, days 1, 2 and 7 after admission and at a 1-month follow-up visit. Plasma concentrations of PAI-1 were measured using a sandwich ELISA kit. The PAI-1 4G/5G polymorphism was typed by allele-specific PCR analysis.ResultsConcentrations of PAI-1 on admission and peak values of PAI-1 during admission were higher than the values measured in healthy controls. Survival was significantly worse in patients with PAI-1 concentrations in the highest tertile (at admission: OR 4.7 [95% CI 0.9–23.8], peak value during admission: OR 6.3 [95%CI 1.3–30.8]). No association was found between the PAI-1 4G/5G polymorphism, and PAI-1 plasma concentrations, dengue disease severity and mortality from dengue.ConclusionThese data suggest that the 4G/5G polymorphism has no significant influence on PAI-1 concentrations in dengue virus infected patients and is not associated with the risk of death from dengue. Other factors contributing to the variability of PAI-1 plasma concentrations in patients with dengue need to be explored.

Chronic renal failure is accompanied by endothelial activation and a large increase in microparticle numbers with reduced procoagulant capacity

BACKGROUND In patients with chronic renal failure (CRF), cardiovascular disease is the leading cause of increased morbidity and mortality. We hypothesized a role for endothelial activation and microparticle (MP) numbers and procoagulant activity in the pre-thrombotic state of these patients. METHODS We analysed blood samples of 27 patients with CRF [8 chronic kidney disease Stage 4 (CKD4), 9 peritoneal dialysis (PD) and 10 haemodialysis (HD), samples taken before and after HD] and 10 controls. Degree and nature of endothelial activation were assessed by measuring mature von Willebrand factor (vWF) and vWF propeptide levels. Cellular MPs were characterized by flow cytometry and MP-specific thrombin generation (TG) measurements. RESULTS CRF was accompanied by chronic (CKD4 and PD) or acute (HD) endothelial activation. Patients with CRF had substantially higher MP numbers than controls (median 9400 versus 4350×10(6)/L, P=0.001), without significant differences between the treatment subgroups or between pre- and post-HD. The vast majority of MPs were platelet derived. Of the minor populations, endothelial MPs and tissue factor-bearing MPs were more abundant in CRF. MPs were procoagulant, but the increase in numbers was not reflected in a proportional increase in MP-specific TG. CONCLUSION Renal failure is accompanied by endothelial activation of a different nature in CKD4 and PD patients compared to HD patients, and results in all subgroups in an increase of mainly platelet-derived MPs that appear to be less procoagulant than in other disease states, possibly because of the uraemic functional defect of their cellular source.

Elevated numbers and altered subsets of procoagulant microparticles in breast cancer patients using endocrine therapy.

INTRODUCTION Microparticles (MP) can be elevated in cancer and thromboembolic disease. We hypothesized a role for MP in the hypercoagulable state in breast cancer patients using endocrine therapy, in whom both cancer and the use of endocrine therapy are independent risk factors for the development of thrombosis. DESIGN AND METHODS Plasma samples were collected from 40 breast cancer patients using endocrine therapy (20 patients without metastases receiving adjuvant therapy and 20 patients with metastatic disease treated in a palliative setting) and from 20 female healthy controls. The endocrine therapy used was either an anti-estrogen or an aromatase inhibitor. Numbers and cellular origin of MP subsets were analyzed by flowcytometry. MP-associated procoagulant activity was measured using a thrombin generation assay using conditions that allow analysis of MP induced thrombin generation. RESULTS Breast cancer patients using endocrine therapy had higher levels of MP positive for Annexin V (median 10000 vs 6500×10E6/l), P-selectin (330 vs 200×10E6/l), tissue factor (33 vs 15×10E6/l), and of MP derived from platelets (CD41) and leukocytes (CD45). Thrombin generation in plasma was dependent on the presence of MP and thrombin generation performed after addition of isolated MP to normal plasma showed a higher endogenous thrombin potential (1105 vs 1029 nM.min) in breast cancer patients. No differences were observed in MP levels and thrombin generation parameters between the metastatic and adjuvant group. CONCLUSION Breast cancer patients using endocrine therapy have an increased MP number and a higher MP-dependent thrombin generation, irrespective of the presence of metastatic disease. Altered MP subset characteristics in these patients, especially the higher number of (activated) platelet derived MP and leukocyte derived MP, may in part explain a heightened procoagulant state in breast cancer patients using endocrine therapy.

Clinical performance evaluation of the CellaVision Image Capture System in the white blood cell differential on peripheral blood smears

Introduction Differential counting and morphological analysis of peripheral blood smears is of great diagnostic importance to the clinician. For economic and time-saving reasons, automated imaging processes have been successfully introduced over the years. The aim of this study was to investigate whether a new morphology system, the CellaVision Image Capture System (CICS), can be used to perform a white blood cell (WBC) differential on peripheral blood smears. Methods WBCs in 200 peripheral blood smears were analysed with the CICS method and compared with the DM96 method to establish accuracy, short-term imprecision and clinical sensitivity and specificity for morphology. For establishing long-term imprecision, two blood smears were analysed for 20 days with the CICS method. Results Evaluation of accuracy in 199 samples demonstrated a good correlation for the CICS when compared with the postclassification on the DM96. Regression coefficients ranged from 0.97 for monocyte counts to 0.99 for neutrophil counts. All regression lines showed slopes with 1 and intercepts with 0 within the 95% CI. Long-term imprecision was less than 5% for neutrophils, eosinophils, basophils, lymphocytes and monocytes. Comparison of the short-term imprecision demonstrated that the SD did not differ by more than 1.1% between the DM96 method and the CICS method. Clinical sensitivity of the CICS was 93.5% and specificity was 97.8%. Specificity and sensitivity for blasts was 100%. Conclusions The CICS has proven to be a reliable and accurate tool in the differential WBC count on peripheral blood smears and provides small laboratories with a 24 h available real-time digital differential WBC count on peripheral blood smears and consultation for patients in remote locations.