Plasma levels of platelet-derived microvesicles are associated with risk of future venous thromboembolism

Background - Microvesicles (MVs) are small double‐membrane encapsulated particles shed from cells. Case‐control studies have reported elevated plasma levels of platelet‐derived MVs (PDMVs) in patients with venous thromboembolism (VTE). However, it is not known whether high PDMV levels is a risk factor or a consequence of the acute VTE event. Objectives - To investigate the association between PDMVs in plasma and risk of future incident VTE. Methods - We performed a population‐based nested case‐control study with 314 VTE cases and 705 age‐ and sex‐matched controls (from The Tromsø Study) to investigate the association between the proportion of PDMVs (PDMVs%) in plasma and risk of future incident VTE. MVs isolated from plasma sampled at baseline (i.e., before VTE) were stained for platelet markers and analyzed by flow cytometry. PDMVs% were defined as the number of PDMVs divided by the total number of MVs. Odds ratios (ORs) with 95% confidence intervals (CI) for VTE risk were estimated across quartiles of PDMVs%. Results - Subjects with PDMVs% in the highest quartile had an OR for VTE of 1.78 (95% CI: 1.21–2.64) and 1.99 (95% CI: 1.24–3.26) for provoked VTE, compared to those in the lowest quartile. The association was moderately affected by multivariable adjustment for age, sex, body mass index, C‐reactive protein, platelet count, and cancer. The OR for VTE was higher when the time between blood sampling and event was shorter. Conclusions - Our results show that high proportions of PDMVs are associated with future risk of incident VTE and imply a role of platelet activation in the pathogenesis of VTE

Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

Peer reviewe

Plasma lipopolysaccharide-binding protein is a biomarker for future venous thromboembolism: Results from discovery and validation studies

Background Effect-size underestimation impedes biomarker identification. Long follow-up time in prospective studies attenuates effect-size estimates for transient biomarkers, while disease category–specific biomarkers are affected by merging of categories. Venous thromboembolism (VTE) encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE). Objectives (i) To re-analyze untargeted proteomic data to identify biomarker candidates for future VTE that differ between DVT and PE and are attenuated by extended time between sampling and VTE. (ii) To perform targeted candidate validation. Patients/Methods A VTE case-control discovery study and a nested case-control validation study were derived from the general population surveyed in 1994–95. Plasma was obtained at study enrollment, and VTE events were registered until 2007. Untargeted proteomic data were re-analyzed for candidate discovery. Lipopolysaccharide-binding protein (LBP) was validated by enzyme-linked immunosorbent assay. Results Elevated LBP was discovered as a candidate DVT biomarker in women with less than 3 years between blood sampling and DVT. In the validation study, the odds ratio (OR) for DVT was 2.03 (95% confidence intervals [CI]: 1.53–2.74) per standard deviation (SD) increase in LBP for women with less than 3 years between blood sampling and DVT. Adjustment for age, body mass index, and C-reactive protein attenuated the OR to 1.79 (95% CI: 1.25–2.62) per SD. In the validation study, we observed an OR for VTE of 0.47 (95% CI: 0.28–0.77) for men in the 25th to 50th percentiles when compared to the lowest quartile. Conclusions We discovered and validated increased LBP as a predictive biomarker for DVT in women. We found an increased VTE risk for men in the lowest quartile of LBP

A rapid, sensitive, and specific assay to measure TF activity based on chromogenic determination of thrombin generation

Background - Most tissue factor (TF) activity assays are based on measurement of factor X (FX) activation by TF in the presence of factor VII (FVII)/FVIIa. This requires long incubation, which may result in TF-independent activity of FX and inaccurate measurement of TF activity. Aim - To develop a sensitive and specific TF activity assay, which does not register a non-specific TF activity, using commercial coagulation factors. Methods - Tissue factor activity was measured based on the ability of TF to accelerate the activation of FX by FVIIa in the presence of factor V (FV)/Va, prothrombin, and phospholipids. Following 4 min incubation at 37°C, TF activity was quantified in test samples of different nature by thrombin generation using a chromogenic substrate. Results - The TF activity assay proved high sensitivity (low fM range) and specificity, assessed by neutralization of TF activity by anti-TF antibody and the use of FVIIai. TF activity was detected in extracellular vesicles (EVs) derived from HAP1-TF+cells, while no activity was measured in EVs from HAP1-TF/KO cells. The assay was applicable for measurement of TF activity on the surface of live endothelial cells and monocytes activated in vitro, and cell lysates. Infusion of low dose lipopolysaccharide (2 ng/kg bodyweight endotoxin) caused a transient 8-fold increase (peaked at 4 h) in TF activity in EVs isolated from plasma of healthy volunteers. Conclusion - Our assay provides a fast, sensitive, and specific measurement of TF activity. It reliably quantifies TF activity on cell surface, cell lysate, and isolated EVs. The assay can be used for laboratory and clinical research

A modified clot-based assay to measure negatively charged procoagulant phospholipids

Growing evidence supports a role for extracellular vesicles (EVs) in haemostasis and thrombosis due to exposure of negatively charged procoagulant phospholipids (PPL). Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF+ EVs

Plasma procoagulant phospholipid clotting time and venous thromboembolism risk

Background - Negatively charged procoagulant phospholipids, phosphatidylserine (PS) in particular, are vital to coagulation and expressed on the surface membrane of extracellular vesicles. No previous study has investigated the association between plasma procoagulant phospholipid clotting time (PPLCT) and future risk of venous thromboembolism (VTE). Objectives - To investigate the association between plasma PPLCT and the risk of incident VTE in a nested case-control study. Methods - We conducted a nested case-control study in 296 VTE patients and 674 age- and sex-matched controls derived from a general population cohort (The Tromsø Study 1994–2007). PPLCT was measured in platelet-free plasma using a modified factor Xa-dependent clotting assay. Logistic regression was used to estimate odds ratio (OR) with 95% confidence intervals (CI) for VTE with PPLCT modelled as a continuous variable across quartiles and in dichotomized analyses. Results - There was a weak inverse association between plasma PPLCT and risk of VTE per 1 standard deviation increase of PPLCT (OR 0.93, 95% CI 0.80–1.07) and when comparing those with PPLCT in the highest quartile (OR 0.89, 95% CI 0.60–1.30) with those in the lowest quartile. Subjects with PPLCT >95th percentile had substantially lowered OR for VTE (OR 0.35, 95% CI 0.13–0.81). The inverse association was stronger when the analyses were restricted to samples taken shortly before the event. The risk estimates by categories of plasma PPLCT were similar for deep vein thrombosis and pulmonary embolism. Conclusion - Our findings suggest that high plasma PPLCT is associated with reduced risk of VTE

Impact of preanalytical conditions on plasma concentration and size distribution of extracellular vesicles using Nanoparticle Tracking Analysis

Optimal pre-analytical handling is essential for valid measurements of plasma concentration and size distribution of extracellular vesicles (EVs). We investigated the impact of plasma preparation, various anticoagulants (Citrate, EDTA, CTAD, Heparin), and fasting status on concentration and size distribution of EVs measured by Nanoparticle Tracking Analysis (NTA). Blood was drawn from 10 healthy volunteers to investigate the impact of plasma preparation and anticoagulants, and from 40 individuals from a population-based study to investigate the impact of postprandial lipidemia. Plasma concentration of EVs was measured by NTA after isolation by high-speed centrifugation, and size distribution of EVs was determined using NTA and scanning electron microscopy (SEM). Plasma concentrations and size distributions of EVs were essentially similar for the various anticoagulants. Transmission electron microscopy (TEM) confrmed the presence of EVs. TEM and SEM-analyses showed that the EVs retained spherical morphology after high-speed centrifugation. Plasma EVs were not changed in postprandial lipidemia, but the mean sizes of VLDL particles were increased and interfered with EV measurements (explained 66% of the variation in EVs-concentration in the postprandial phase). Optimization of procedures for separating VLDL particles and EVs is therefore needed before NTAassessment of EVs can be used as biomarkers of disease

Associations between complement pathways activity, mannose-binding lectin, and odds of unprovoked venous thromboembolism

INTRODUCTION: Deep vein thrombosis (DVT) originates in the valvular sinuses of large veins in a local milieu characterized by stasis and severe hypoxia. This may induce complement- and coagulation activation, which potentially increases the risk of venous thromboembolism (VTE). The aim of the present study was to investigate whether the activity of the complement pathways, the level of mannose-binding lectin (MBL) and tissue-factor (TF) induced thrombin generation were associated with risk of unprovoked VTE. METHODS: A case-control study was performed in patients with unprovoked VTE (n = 24) and age- and sex-matched healthy controls (n = 24). Serum complement pathway activity was measured by the total complement screen assay (Wieslab®). MBL was quantified by ELISA. Plasma TF-induced thrombin generation was measured using the CAT-assay. RESULTS: Activity in the highest quintile of the classical pathway was associated with increased odds of unprovoked VTE (OR 4.5, 95% CI; 0.8-24.7). Moreover, MBL deficiency (≤100 ng/ml) was associated with unprovoked VTE (OR 3.5, 95% Cl; 0.8-15.3). VTE patients had shortened TF-induced lag-time (4.8 ± 0.6 min vs. 5.8 ± 2.1 min, p < 0.001) and a higher endogenous thrombin potential (ETP) (1383 ± 267 nM∗h vs. 1265 ± 247 nM∗h, p = 0.07) than controls. No association between the classical complement pathway activity or MBL deficiency, and parameters of TF-induced thrombin generation was observed. CONCLUSION: Our findings suggest that high activity of the classical complement pathway, and MBL deficiency, might be associated with an increased odds of unprovoked VTE, independent of activation of TF-induced coagulation

Plasma levels of growth differentiation factor 15 are associated with future risk of venous thromboembolism

Growth differentiation factor 15 (GDF-15), a marker of inflammation and oxidative stress, has emerged as a biomarker for arterial cardiovascular disease. However, the association between GDF-15 and venous thromboembolism (VTE) remains uncertain. We therefore investigated the association between plasma GDF-15 levels and future risk of incident VTE and explored the potential of a causal association using Mendelian randomization (MR). We conducted a population-based nested case-control study comprising 416 VTE patients and 848 age- and sex-matched controls derived from the Tromsø Study. Logistic regression was used to calculate odds ratios (ORs) for VTE across GDF-15 quartiles. For the MR, we used data from the International Network on Venous Thrombosis (INVENT) consortium to examine whether single nucleotide polymorphisms (SNPs) associated with GDF-15 levels with genome-wide significance were related to VTE. We found that the ORs for VTE increased across GDF-15 quartiles (Ptrend = .002). Participants with GDF-15 values in the highest quartile (≥358 pg/mL) had an OR for VTE of 2.05 (95% confidence interval, 1.37-3.08) compared with those with GDF-15 in the lowest quartile (<200 pg/mL) in the age- and sex-adjusted model. ORs remained essentially the same after further adjustment for body mass index, smoking, hormone therapy, physical activity, and C-reactive protein. Similar results were obtained for provoked/unprovoked events, deep vein thrombosis, and pulmonary embolism. GDF-15 levels, as predicted by the SNPs, were not associated with VTE in MR. Our results indicate that high GDF-15 levels are associated with increased risk of VTE, but MR suggests that this association is not causal

Effect of lower‐leg trauma and knee arthroscopy on procoagulant phospholipid‐dependent activity

Abstract Background Lower‐leg injury and knee arthroscopy are both associated with venous thromboembolism (VTE). The mechanism of VTE in both situations is unknown, including the role of procoagulant microparticles. This may provide useful information for individualizing thromboprophylactic treatment in both patient groups. Objective We aimed to study the effect of (1) lower‐leg trauma and (2) knee arthroscopy on procoagulant phospholipid‐dependent (PPL) activity plasma levels. Methods POT‐(K)CAST trial participants who did not develop VTE were randomly selected for the current study. Plasma was collected shortly after lower‐leg trauma or before and after knee arthroscopy. For aim 1, samples of 67 patients with lower‐leg injury were compared with control samples (preoperative samples of 74 patients undergoing arthroscopy). Linear regression was used to obtain mean ratios (natural logarithm retransformed data), adjusted for age, sex, body mass index, infections, and comorbidities. For aim 2, pre‐ and postoperative samples of 49 patients undergoing arthroscopy were compared using paired t tests. PPL activity was measured using modified activated factor X–dependent PPL clotting assay. Results For aim 1, PPL activity levels were almost threefold higher in patients with lower‐leg injury compared with controls, that is, mean ratio, 2.82 (95% confidence interval [CI], 1.98‐4.03). For aim 2, postoperative PPL activity levels did not change significantly, that is, mean change, −0.72 mU/mL (95% CI, −2.03 to 0.59). Conclusion Lower‐leg trauma was associated with increased plasma levels of PPL activity, in contrast to knee arthroscopy. Lower‐leg trauma triggers the release of procoagulant microparticles