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

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.

Rosuvastatin treatment decreases plasma procoagulant phospholipid activity after a VTE: A randomized controlled trial.

BACKGROUND: Venous thromboembolism (VTE) is a frequent cardiovascular disease with severe complications, including recurrence and death. There is a great need for alternative prophylactic treatment options as anticoagulation is accompanied by increased bleeding risk. Statins are reported to reduce the risk of incident and recurrent VTE, but the mechanisms are elusive. Procoagulant phospholipids (PPL), and phosphatidylserine in particular, are crucial for efficient coagulation activation, but no studies have investigated the effect of statin treatment on plasma PPL activity. OBJECTIVES: To investigate the impact of rosuvastatin treatment on plasma PPL activity and levels of extracellular vesicles (EVs). PATIENTS/METHODS: Patients with a history of VTE (≥18 years) allowed to stop anticoagulant treatment were randomized to either 20 mg/day of rosuvastatin treatment or no treatment for 28 days in the Statins Reduce Thrombophilia (NCT01613794) trial. Plasma samples were collected at baseline and study end. PPL activity was measured in samples from 245 participants using a factor Xa-dependent clotting assay and EV levels by flow cytometry. RESULTS: Rosuvastatin treatment yielded an overall 22% (95% confidence interval [CI] -38.2 to -5.8) reduction in PPL activity, and 37% (95% CI -62.9 to -11.2) reduction in PPL activity in participants with a history of pulmonary embolism. The effect of rosuvastatin on plasma PPL activity was not explained by changes in total cholesterol nor change in levels of total- or platelet-derived EVs. CONCLUSIONS: Rosuvastatin treatment caused a substantial decrease in plasma PPL activity, suggesting that a PPL-dependent attenuation of coagulation activation may contribute to a reduced VTE risk following statin treatment.

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) confirmed 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 NTA-assessment of EVs can be used as biomarkers of disease.