Prospective and comparative study of paroxysmal nocturnal hemoglobinuria patients treated or not by eculizumab: Focus on platelet extracellular vesicles.

Thrombosis are severe complications of paroxysmal nocturnal hemoglobinuria (PNH), effectively reduced by eculizumab. Extracellular vesicles (EVs) may play a central role. The objective of this study was to assess the procoagulant activity of plasma isolated from PNH patients (treated or not by eculizumab) and to quantify their circulating EVs.We iteratively collected the platelet-free-plasma of 17 PNH patients and 16 matched healthy volunteers, quantified their circulating EVs by flow cytometry and evaluated their procoagulant activity by thrombin generation and STA-Procoag-procoagulant phospholipid (PPL) assays.A significant decrease of EVs from platelets (P = .024) and an increase of the STA-Procoag-PPL clotting time (P = .049) was observed after initiation of eculizumab and up to 11 weeks after. This reduction of prothrombotic biomarkers was not observed with the thrombin generation test due to a lack of sensitivity of this assay. Active hemolysis was observed in 90% of patients and elevated D-dimers in 41% of them. However, no significant difference was observed between patients and control subjects regarding the procoagulant activity, the EVs quantity, or the cellular origin. Lactate dehydrogenase (LDH) levels were lower in eculizumab-treated patients compared to nontreated patients (441 vs 2448 IU/L). D-dimers and LDH decreased after administration of eculizumab (mean decrease of 1307 ng/mL and 4159 IU/L, respectively).These observations suggest a decrease of the phospholipid-dependent procoagulant potential of EVs after eculizumab therapy in PNH patients. TRIAL REGISTRATION:: NUB: B039201214365.

Optimal wavelength for the clot waveform analysis: Determination of the best resolution with minimal interference of the reagents.

INTRODUCTION: Clot waveform analysis (CWA), a new methodology to assess coagulation process, can be usefully applied in various clinical settings. However, its clinical use is limited mainly because of the absence of standardization. No consensus exists regarding the wavelengths at which CWA has to be performed what is crucial for the sensitivity of the CWA. OBJECTIVES: The primary aim of this study is to determine which wavelength is the most sensitive and specific for CWA. Interindividual baseline absorbance will also be assessed as the impact of reagents from the intrinsic, extrinsic, and common coagulation pathway will be determined. METHODS: Plasma samples were screened at wavelengths from 280 to 700 nm to provide absorbance spectra in clotted and nonclotted plasma. The interindividual variability of baseline absorbance was obtained by screening plasma from 50 healthy individuals at 340, 635, and 671 nm. The inner-filter effect of reagents was assessed in plasma or serum when appropriate at the same wavelengths. The reagents were those commonly used for activated partial thromboplastin time, prothrombin time, thrombin time, and dilute Russell's viper venom time. RESULTS: Clotted plasma has higher absorbance value than nonclotted plasma (P < 0.01). The absorbance of all type of samples is higher at 340 nm than at >600 nm (P < 0.01). The interindividual variability at the different wavelengths was around 25%. However, except with the STA®-CKPrest® and STA®-NeoPTimal®, the reagents do not have a significant effect on the baseline absorbance. CONCLUSIONS: Wavelengths above 650 nm are recommended to perform CWA. Most of the commercialized reagents can be used for CWA.

Betrixaban: Impact on Routine and Specific Coagulation Assays-A Practical Laboratory Guide.

INTRODUCTION: Betrixaban is a novel direct oral factor Xa inhibitor approved by the Food and Drug Administration for prophylaxis of venous thromboembolism in adult patients hospitalized for an acute illness at risk for thromboembolic complications. Assessment of the anti-coagulant effect of betrixaban may be useful in some situations. Also, clinicians need to know how routine coagulation assays are influenced. OBJECTIVE: The aim of this study is to determine which coagulation assay(s) should be used to assess the impact of betrixaban on haemostasis and provide laboratory guidance for their interpretation. MATERIALS AND METHODS: Betrixaban was spiked at final concentrations ranging from 0 to 250 ng/mL in platelet-poor plasma. Different reagents from several manufacturers were tested and the impact of betrixaban on pro-thrombin time (PT), activated partial thromboplastin time (aPTT), dilute Russel viper venom time (dRVV-T), chromogenic anti-Xa assays, thrombin generation assay (TGA), and a large panel of haemostasis diagnostic tests has been assessed. RESULTS: A concentration-dependent prolongation of aPTT, PT and dRVV-T is observed. The sensitivity mainly depends on the reagent. Chromogenic anti-Xa assays show high sensitivity depending on the reagent and/or the methodology. These assays applicable for other direct factor Xa inhibitors have to be adapted to obtain a relevant range of measurement. TGA may also be attractive to assess the anti-coagulant activity of betrixaban. CONCLUSION: Adapted chromogenic anti-Xa assays are the most appropriate assays to estimate the concentration of betrixaban. Betrixaban significantly affects several haemostasis diagnostic tests and this needs to be taken into consideration when requesting and interpreting such tests.

Application of a clot-based assay to measure the procoagulant activity of stored allogeneic red blood cell concentrates.

BACKGROUND: Thrombotic effects are possible complications of red blood cell transfusion. The generation and accumulation of procoagulant red blood cell extracellular vesicles during storage may play an important role in these thrombotic effects. The objective of this study was to assess the value of a simple phospholipid-dependent clot-based assay (STA®-Procoag-PPL) to estimate the procoagulant activity of stored red blood cells and changes in this activity during storage of the blood component. MATERIALS AND METHODS: Extracellular vesicles from 12 red blood cell concentrates were isolated at 13 storage time-points and characterised by quantitative and functional methods: the degree of haemolysis (direct spectrophotometry), the quantification and determination of cellular origin (flow cytometry) and the procoagulant activity (thrombin generation and STA®-Procoag-PPL assays) were assessed. RESULTS: The mean clotting time of extracellular vesicles isolated from red blood cell concentrates decreased from 117.2±3.6 sec on the day of collection to 33.8±1.3 sec at the end of the storage period. This illustrates the phospholipid-dependent procoagulant activity of these extracellular vesicles, as confirmed by thrombin generation. Results of the peak of thrombin and the STA®-Procoag-PPL were well correlated (partial r=-0.41. p<0.001). In parallel, an exponential increase of the number of red blood cell-derived extracellular vesicles from 1,779/µL to 218,451/µL was observed. DISCUSSION: The STA®-Procoag-PPL is a potentially useful technique for assessing the procoagulant activity of a red blood cell concentrate.

Procoagulant activity of extracellular vesicles as a potential biomarker for risk of thrombosis and DIC in patients with acute leukaemia.

Haemostatic complication is common for patients with hematologic malignancies. Recent studies suggest that the procoagulant activity (PCA) of extracellular vesicles (EV) may play a major role in venous thromboembolism and disseminated intravascular coagulation (DIC) in acute leukaemia. To study the impact of EVs from leukaemic patients on thrombin generation and to assess EV-PCA as a potential biomarker for thrombotic complications in patients with acute leukaemia. Blood samples from a cohort of patients with newly diagnosed acute leukaemia were obtained before treatment (D-0), 3 and 7 days after treatment (D-3 and D-7). Extracellular vesicles were isolated and concentrated by ultracentrifugation. EV-PCA was assessed by thrombin generation assay, and EV-associated tissue factor activity was measured using a commercial bio-immunoassay (Zymuphen MP-TF®). Of the 53 patients, 6 had increased EV-PCA at D-0 and 4 had a thrombotic event. Patients without thrombotic events (n = 47) had no elevated EV-PCA. One patient had increased EVs with procoagulant activity at D-3 and developed a DIC at D-5. This patient had no increased EVs-related tissue factor activity from D-0 to D-7 (<2 pg/ml). Eight patients had increased EVs with tissue factor activity (>2 pg/ml), of these, four had a thrombosis and two had haemorrhages. Procoagulant activity of extracellular vesicles could have a predictive value in excluding the risk of thrombotic events. Our findings also suggest a possible association between thrombotic events and EV-PCA.

Effect of leukocyte- and platelet-rich fibrin (L-PRF) on bone regeneration: a study in rabbits.

BACKGROUND: The positive effect of leukocyte- and platelet-rich fibrin (L-PRF) on osteogenesis has been widely described in vitro. However, clinical and preclinical studies are very little and controversial in demonstrating a significant beneficial effect of L-PRF in bone regeneration. PURPOSE: The goal of the present study was to compare the potential effect of L-PRF in a standardized model. MATERIALS AND METHODS: A total of 72 hemispheres were implanted on the calvaria of 18 rabbits and filled with three different space fillers: L-PRF, bovine hydroxyapatite (BHA), BHA + L-PRF, and an empty hemisphere was used as control. Six rabbits were sacrificed at three distinct time points: 1 week, 5 weeks, and 12 weeks. Histological and histomorphometrical analyses were carried out. RESULTS: At the early phase of bone regeneration (1 week), from a descriptive analysis, a higher proportion of connective tissue colonized the regeneration chamber in the two groups containing BHA particles. Nevertheless, no statistical differences were found within the four groups in terms of bone quantity and quality at each timepoint (p = .3623). CONCLUSIONS: According to the present study, L-PRF does not seem to provide any additional effect on the kinetics, quality, and quantity of bone in the present model of guided bone regeneration.

Contributing role of extracellular vesicles on vascular endothelium haemostatic balance in cancer.

Extracellular vesicles (EVs) generated during tumourigenesis are thought to play a major role in the hypercoagulant state observed in cancer patients. They exhibit negatively charged phospholipids and tissue factor (TF) that promote coagulation cascade activation. In addition, they contain surface proteins and cytoplasmic molecules, both originating from the producing cell that can impact target cells' expression. By targeting endothelial cells of blood vessels, these EVs could disturb the physiological anticoagulant properties of these cells and be partly responsible for the vascular endothelium activation observed in cancer patients. Indeed, vascular endothelium naturally exhibits heparin-like proteoglycan, TF pathway inhibitor and protein C anticoagulant pathway that prevent thrombosis in physiological condition. An overexpression of TF and a decreased expression of coagulation cascade inhibitors have been reported after EVs' treatment of endothelial cells. The induction of apoptosis and an increased expression of platelet adhesion molecules have also been highlighted. These events may promote thrombus formation in cancer. The aim of this paper is to provide a targeted review on the current evidence and knowledge of roles and impact of EVs on endothelial surface anticoagulant and procoagulant factors and cellular adhesion molecules expression.

Microparticle bearing tissue factor: a link between promyelocytic cells and hypercoagulable state.

Patients with hematological malignancies have a 28-fold increased risk of venous thromboembolism (VTE). Among patients with acute myelogenous leukemia (AML), the 2-year cumulative incidence of VTE is 5.2%. Several studies suggest that microvesicles (MVs) harboring TF may play a role in VTE and disseminated intravascular coagulation (DIC) in acute promyelocytic leukemia (APL). The aim of this study was to assess the capacity of untreated (APL) cells to shed procoagulant MVs. APL cells (NB4 and HL-60 cell lines) and MVs were separated by filtration (0.1-0.22-0.45-0.65 µm). The procoagulant activity (PCA) was assessed by thrombin generation assay (TGA). Alternatively, MVs were incubated with anti-Tissue Factor (TF) antibodies, with annexin V to assess the contribution of TF and phospholipids (PL) to the PCA, respectively. NB4 cells had a high PCA mainly triggered by MVs of size under 0.45 µm. The PCA of MVs was related to the expression of active TF and PL. HL-60 cells had a weaker PCA since TF is mostly present in its inactive form. Moreover, HL-60 do not produce MVs<0.65 µm associated with PCA. MVs could have a predicting value for VTE and DIC in patients with acute promyelocytic leukemia and could inform physicians about the optimal use of a thromboprophylaxis.

Characterisation of tissue factor-bearing extracellular vesicles with AFM: comparison of air-tapping-mode AFM and liquid Peak Force AFM.

INTRODUCTION: Extracellular vesicles (EVs) are shed from cells and carry markers of the parent cells. Vesicles derived from cancer cells reach the bloodstream and locally influence important physiological processes. It has been previously shown that procoagulant vesicles are circulating in patients' fluids. These EVs are therefore considered as promising biomarkers for the thrombotic risk. Because of their small size, classical methods such as flow cytometry suffer from limitation for their characterisation. Atomic force microscopy (AFM) has been proposed as a promising complementary method for the characterisation of EVs. OBJECTIVES: THE OBJECTIVES OF THIS STUDY ARE: (a) to develop and validate AFM with specific antibodies (anti-TF) and (b) to compare air and liquid modes for EVs' size and number determination as potential biomarkers of the prothrombotic risk. METHODS: AFM multimode nanoscope III was used for air tapping mode (TM). AFM catalyst was used for liquid Peak Force Tapping (PFT) mode. Vesicles are generated according to Davila et al.'s protocol. Substrates are coated with various concentrations of antibodies, thanks to ethanolamine and glutaraldehyde. RESULTS: Vesicles were immobilised on antibody-coated surfaces to select tissue factor (TF)-positive vesicles. The size range of vesicles observed in liquid PFT mode is 6-10 times higher than in air mode. This corresponds to the data found in the literature. CONCLUSION: We recommend liquid PFT mode to analyse vesicles on 5 µg/ml antibody-coated substrates.

Thrombin generation assay and transmission electron microscopy: a useful combination to study tissue factor-bearing microvesicles.

INTRODUCTION: Patients with cancer have a 7- to 10-fold increased risk of developing venous thromboembolism. Circulating microvesicles could be a useful predictive biomarker for venous thromboembolism in cancer. Validated and standardised techniques that could be used to determine the complete microvesicle phenotype are required. OBJECTIVES: These were two-fold: a) to characterise tissue factor (TF)-bearing microvesicles released by cultured breast cancer cells MDA-MB-231 by flow cytometry (FCM), transmission electron microscopy (TEM) and thrombin generation assay (TGA); and b) to validate the sensitivity and variability intra/inter-assay of TGA as a useful method to study the procoagulant activity (PCA) of microvesicles. METHODS: Cultured breast cancer cells MDA-MB-231 were incubated for 45 minutes at 37°C. Samples were then centrifuged or not at 4,500 g for 15 minutes, and cells and MVs or MV-containing supernatants were used for TEM, FCM and TGA. In activity assays, microvesicles (i.e. cell-depleted supernatants) were incubated with anti-TF antibodies or with annexin V to assess the contribution of TF and phospholipids to the PCA. Alternatively, supernatants were filtered through 0.1, 0.22, 0.45 or 0.65 µm membranes and subjected to TGA. RESULTS: The majority of the PCA was associated with microvesicles smaller than 0.1 µm, and the mean microvesicle size estimated by TEM after 10,000 g centrifugation was 121±54 nm with a majority of vesicles between 100 and 200 nm. Microvesicles derived from 5,000 MDA-MB-231cells/ml were sufficient to significantly increase the thrombin generation of normal pooled plasma. CONCLUSIONS: TEM, FCM and filtration coupled to TGA represent a useful combination to study the PCA of TF-bearing microvesicles, whatever their size. And it will be interesting to implement these techniques in patients.

Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles.

Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. 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 TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.