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

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

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