| Literature DB >> 30640949 |
Thøger Nielsen1,2,3, Søren Risom Kristensen1,2,3, Henrik Gregersen2,3,4, Elena Manuela Teodorescu2,3,4, Gunna Christiansen5, Shona Pedersen1,2,3.
Abstract
Multiple myeloma (MM) patients have increased risk of developing venous thromboembolism, but the underlying mechanisms and the effect on the coagulation system of the disease and the current cancer therapies are not known. It is possible that cancer-associated extracellular vesicles (EV), carrying tissue factor (TF) and procoagulant phospholipids (PPL) may play a role in thrombogenesis. The aim of this study was to perform an in-depth analysis of procoagulant activity of small and large EVs isolated from 20 MM patients at diagnosis and after receiving first-line treatment compared with 20 healthy control subjects. Differential ultracentrifugation at 20,000 × g and 100,000 × g were used to isolate EVs for quantitative and phenotypical analysis through nanoparticle tracking analysis, Western blotting and transmission electron microscopy. The isolated EVs were analyzed for procoagulant activity using the calibrated automated thrombogram technique, a factor Xa-based activity assay, and the STA Procoag-PPL assay. In general, MM patients contained more EVs, and immunoelectron microscopy confirmed the presence of CD9- and CD38-positive EVs. EVs in the 20,000 × g pellets from MM patients exerted procoagulant activity visualized by increased thrombin generation and both TF and PPL activity. This effect diminished during treatment, with the most prominent effect observed in the high-dose chemotherapy eligible patients after induction therapy with bortezomib, cyclophosphamide, and dexamethasone. In conclusion, the EVs in patients with MM carrying TF and PPL are thus capable of exerting procoagulant activity.Entities:
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Year: 2019 PMID: 30640949 PMCID: PMC6331130 DOI: 10.1371/journal.pone.0210835
Source DB: PubMed Journal: PLoS One ISSN: 1932-6203 Impact factor: 3.240
Characteristics of the multiple myeloma patients at diagnosis.
| Multiple | ||
|---|---|---|
| Number of patients | 20 | |
| Age, years | 70 ± 10 | |
| Male percentage | 55% | |
| ISS stage | ||
| I | 2 (10%) | |
| II | 7 (35%) | |
| III | 11 (55%) | |
| M-protein, g/L | 41.5 ± 19.9 | |
| IgG, | 14 (70%) | |
| kappa | ||
| lambda | ||
| IgA, | 6 (30%) | |
| kappa | ||
| lambda | ||
| INR | 1.1 ± 0.2 | |
| APTT, s | 30 ± 4 | |
| Fibrinogen, μmol/L | 9.4 ± 3.3 | |
| D-dimer, mg/L | 0.80 ± 0.27 | |
| Antithrombin, ×E9 IU/L | 0.88 ± 0.17 | |
| Factor VIII, U/mL | 1.60 ± 0.73 | |
| Protein C, U/mL | 1.10 ± 0.39 | |
| Creatinine, μmol/L | 89 ± 23 / | |
| Carbamide, mmol/L | 7.0 ± 2.6 / | |
| Pt-estimated GFR, mL/min | 74 ± 17 | |
| κ-chain, free, mg/L | 1153.1 ± 3723.9 | |
| λ-chain, free, mg/L | 299.8 ± 697.1 | |
| Calcium, mmol/L | 2.48 ± 0.15 | |
| CRP, mg/L | 7.5 ± 22.7 | |
| Albumin, g/L | 30 ± 4 | |
| Protein, g/L | 106 ± 18 | |
| ALAT, U/L | 23 ± 10 | |
| Haemoglobin, mmol/L | 6.7 ± 1.5 / | |
| Erythrocytes, ×E12/L | 3.44 ± 0.80 / | |
| Platelets, ×E9/L | 198 ± 57 / | |
| Leukocytes, ×E9/L | 6.3 ± 2.2 |
ISS = international staging system; IgG = immunoglobulin G; IgA = immunoglobulin A; INR = international normalized ratio; APTT = activated partial thromboplastin time; GFR = glomerular filtration rate; CRP = C-reactive protein; ALAT = alanine transaminase.
Demographic characteristics of patients the conventional or induction therapy groups including treatment response.
| Conventional therapy | VCD induction therapy | |
|---|---|---|
| Number of patients | 11 | 5 |
| Age, years | 76 ± 5 | 64 ± 5 |
| Male gender | 55% | 40% |
| I | 1 (9%) | 0 (0%) |
| II | 6 (55%) | 2 (40%) |
| III | 4 (36%) | 3 (60%) |
| VCD | 0 (0%) | 5 (100%) |
| MPV | 10 (91%) | 0 (0%) |
| LEN-DEX | 1 (9%) | 0 (0%) |
| Very good partial response | 3 (28%) | 4 (80%) |
| Partial response | 4 (36%) | 1 (20%) |
| Stable disease | 4 (36%) | 0 (0%) |
| M-protein posttreatment reduction, % | 58 ± 25 | 90 ± 9 |
*Mean ± standard deviation; LEN-DEX = lenalidomide and dexamethasone.
Fig 1Analysis of EV characteristics.
Nanoparticle tracking analysis was performed on each pellet (20K and 100K) for controls and MM patients to determine A) particle concentrations and B) mean particle size. The boxplots depict the median, the 25 and 75 percentiles and the whiskers min to max. *P<0.05; **P<0.01; ****P<0.0001. C) The distribution of particle sizes was grouped into three subgroups (<100 nm, 100–200 nm, and >200 nm). D) The pellet pools were analysed by Western blotting for EV-marker CD9 and ectoenzyme CD38. Equivalent volumes of each pellet pool (20K and 100K) from both controls and MM were loaded on the gels. As expected, tetraspanin CD9 was present in all pellet types but enriched in MM pellets, especially in the 20K pellet pool. CD38 was found in all pellet pools, but most abundant in MM pellets (mostly in the 100K pellet pool). E) Immunoelectron microscopy images of gold immunolabelled CD9+ and CD38+ EVs in pellet pools of control and MM pellets (20K and 100K pellets). Images include scale bars determined with ImageJ software.
Fig 2Analysis of procoagulant activity of EVs in SPP.
A) Thrombograms (mean ± standard deviation) depicting thrombin generation when SPP is ‘spiked’ with isolated EVs from controls and MM patients. The results on individual thrombin generation parameters (ETP, peak height, velocity index, lag time, and time-to-peak) are listed in the table as the means ± standard deviation including P. B) PPL activity measured in clotting time differences in SPP ‘spiked’ with isolated EVs. The boxplots depict the median, the 25 and 75 percentiles and the whiskers min to max and the green line and area represent the reference range (mean ± standard deviation) of the SPP. C) Analysis of MV-associated TF was performed on MV suspensions, and MM patients contained overall more TF than controls. D) Correlation matrix depicting the Pearson’s r for correlations between coagulation and particle analyses for the MM pellets. ****P<0.0001.
Fig 3The procoagulant activity of EVs from 20K pellets of MM patients at diagnosis and after the first-line treatment regimen.
A) (Left, middle) Thrombograms (mean ± standard deviation) depicting the outcome of EV-mediated thrombin generation before and after conventional therapy (HDCT ineligible patients) or a VCD induction therapy (HDCT eligible patients). The results on individual thrombin generation parameters are listed in the table as the means ± standard deviation including P. (Right) The effect of treatment on PPL activity of EVs ‘spiked’ into SPP, **P<0.01. The green line and area represent the reference range (mean ± SD) of the SPP. B) Size distribution (left), particle concentration (middle), and mean size (left) before and after first-line treatment as measured by the means of nanoparticle tracking analysis. All boxplots depict the median, the 25 and 75 percentiles and the whiskers min to max.