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Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis
Thrombosis Research 143 (2016) 118–121
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Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis Dear Editors, Portal vein thrombosis (PVT) is a common complication of hepatocellular carcinoma (HCC) and is associated with a poor prognosis [1]. However, studies on the hypercoagulable state associated with this neoplasm and its correlation with the risk of PVT are lacking. Microparticles (MP), a population of extracellular vesicles ranging in size from 0.1 to 1 μm, are released from the surface of cells by the process of outward membrane budding through a loss of calcium-dependent membrane phospholipid asymmetry and cytoskeletal rearrangement [2–4]. MP have been extensively investigated for their ability to induce coagulation and participate in thrombosis because of the presence of tissue factor (TF) and the exposure of negatively charged phospholipids [2–4]. Thus far, numerous studies have analysed levels of circulating MP in patients presenting diseases with a high risk for thromboembolic complications [3,5], but there is not enough data available in Literature regarding the presence of MP in HCC and their potential prothrombotic role. Our aim was to investigate the presence and cellular origins of circulating MP in plasma of patients with cirrhosis with and without HCC and compare them with a healthy control population and to evaluate the possible contribution of MP in PVT occurrence in HCC. All consecutive patients presenting established cirrhosis with or without HCC admitted to the Department of Gastroenterology of University of Padua from January to September 2013 were enrolled into this study. Severity of cirrhosis was estimated according to the ChildPugh classification and MELD score. A control group of 50 consecutive healthy subjects matched for age (± 3 years), sex and ethnic origins was recruited. Exclusion criteria were: ongoing antiplatelet/anticoagulant treatment, inherited thrombophilia, undergoing antiviral treatment, dialysis and extrahepatic neoplasms. This study was approved by the Ethical Committee of Padua Medical Hospital. Written informed consent was obtained from each patient and control. Both patients with and without HCC were followed up for 12 months after the recruitment for the occurrence of PVT. Fasting whole blood samples (9 mL) were drawn at enrolment by clean venipuncture from an antecubital vein and collected into vacuum tubes containing109 mM trisodium citrate. Platelet-free plasma was prepared within 3 h by double centrifugation (2 × 15 min at 2500 g) at room temperature. Aliquots were immediately frozen and then stored at − 80 °C until use. Samples were thawed by incubation for 5 min in a water bath at 37 °C immediately before testing and analysed only after a single freeze-thaw cycle. Assays were performed at least 1 month after storage at −80 °C [6]. Classical coagulation tests were performed (platelet count, activated partial thromboplastin time and prothrombin time, factor II, fibrinogen,
http://dx.doi.org/10.1016/j.thromres.2016.05.021 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
factor VIII, and antithrombin activity, protein C antigen, chromogenic and coagulometric activities, protein S coagulometric activity and total and free antigens) as previously described [6]. MP analysis was performed on a Cytomics FC500 flow cytometer (Beckman Coulter, Miami Florida), as previously described [6]. MP expressing phosphatidylserine were identified by size and annexin V-fluorescein isothiocyanate (FITC) (Bender MedSystems GmbH, Vienna, Austria) labelling (annexinV-MP). The different populations of MP were measured co-labelling with antibodies against cell-type specific antigens and annexin V. Endothelial-derived MP were identified using CD62E-PC5 (phycoerythrin-cyanin5.1), platelet-derived MP using CD62P-PE (phycoerythrin) (both Beckman Coulter, Miami, Florida); leukocyte MP using CD45-PC5 (BioLegend Europe, The Netherlands); tissue factor-bearing (TF + MP) with CD142-PE and thrombomodulin-bearing MP with CD141-PE - both from BD, Biosciences, Milan, Italy. Isotype controls were used. Procoagulant activity of the MP was measured using the STA®Procoag PPL assay (Diagnostica Stago, Asnieres, France) [7]. MP TF activity was measured by an “in-house” endpoint assay using a curve generated with commercially available Innovin™ (Dade Behering) as standard, as previously reported by other groups [8,9]. Normally distributed variables were summarized as mean (± SD) and Student's t-test was performed, whereas non-normally distributed variables were summarized as medians with interquartile ranges (IQR) and Mann-Whitney U test was conducted. For multiple comparisons Kruskall-Wallis test was used. Spearman's correlation analysis was used to detect significant correlations. A “p” value b 0.05 was considered statistically significant. The relative risk (RR) and the 95% confidence intervals (CI) were ascertained from contingency tables (PASW Statistics 17.0.2 for Windows). Sixty-five adult cirrhotic patients [Child: A 21, B 27, C 17], 33 with HCC (M/F 25/8; mean age 64.5 ± 12.5 years) and 32 without HCC (M/ F 24/8; mean age 56 ± 14.7 years) were prospectively enrolled. Median MELD was 11 [8–14] in patients with HCC and 13 [10−20] in patients without HCC, respectively (p = 0.13). Portal vein thrombosis was detected in 12 (18%) cirrhotic patients, 8 with HCC and 4 without HCC. Fifty adult healthy subjects [M/F 25/25, age 54 ± 8 years] acted as controls. Patients with cirrhosis and HCC had a significantly higher platelets count and significantly lower protein C coagulometric activity and antigen thancirrhotic patients without HCC (Table 1). Patients with cirrhosis and HCC showed significantly higher median plasma levels of annexin V-MP (p b 0.001), endothelial-derived MP (p b 0.001), platelet-derived MP (0 b 0.005), leukocyte-derived MP (p b 0.001), TF + MP (p b 0.001) and thrombomodulin (TM) + MP (p b 0.05) than cirrhotic patients without HCC. Moreover, cirrhosis without HCC showed significantly higher levels of annexin V-MP (p b 0.01), endothelial-derived MP (p b 0.001), platelet-derived MP (p = 0.02), leukocyte-derived MP (p b 0.001), and TM + MP (p b 0.001) compared to healthy controls. No significant difference was detected for TF + MP between healthy controls and cirrhotic patients without HCC (p = 0.9) (Fig. 1). Considering the overall study
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Table 1 Basal characteristics of the study population.
Age, years (mean ± SD) Etiology: alcohol-virus/other, n CHILD A/B/C, n MELD (median [IQR]) αFP, μg/L (median [IQR]) Portal vein thrombosis, n (%) Time of PVT occurrence after enrolment, months (median [IQR]) Platelets/μL (mean ± SD) Protein C Chromogenic activity, % Coagulometric activity, % Antigen, % TACE (n of procedures in n patients) Percutaneous RF (n of procedures in n patients) Laparoscopic RF (n of procedures in n patients) Sorafenib treatment (n of patients)
Cirrhosis with HCC (n = 33)
Cirrhosis without HCC (n = 32)
p
64.5 ± 12.5 30/3 13/12/8 11 [8–14] 4.5 [2.3–9.5] 8 (24) 7 [6–8] 127 ± 77
56.0 ± 14.7 20/12 8/15/9 13 [10–20] 4.0 [2.3–9.3] 4 (12.5) 7 [6–9] 97 ± 54
ns ns ns ns ns b0.01 ns 0.046
45.0 ± 31.6 39.8 ± 27.1 45.9 ± 29.8 24 in 22 patients 8 in 6 patients 6 in 5 patients 0
56.4 ± 29.8 53.4 ± 25.2 60.8 ± 29.5
ns 0.029 0.035
HCC, hepatocellular carcinoma; αFP, alphafetoprotein; MELD, model for end stage liver disease; IQR, interquartile range, PVT, portal vein thrombosis; TACE, trans-arterial chemo-embolization; RF, radiofrequency.
population no statistically significant differences in MP plasma levels were detected among patients in different Child-Pugh classes. Median phospholipid-dependent clotting time (PPL assay) was significantly shorter in patients with cirrhosis and HCC (52.4 [46.2–70.0] sec) than in patients with cirrhosis without HCC (74.3 [67.8–79.6] sec, p = 0.001) and healthy controls (74.3 [67.8–80.1] sec, p = 0.007). This shortening of the PPL clotting time reflects an increase procoagulant activity due to phospholipid presence. No difference in PPL clotting time was detected between cirrhosis without HCC and controls (p = 0.81). In the overall study population, the levels of annexin V-MP were inversely correlated with PPL clotting time (r = − 0.66, p = 0.002). Median MP TF activity was higher in patients with concomitant cirrhosis and HCC (1.12 [0.9–1.62] pg/mL) than in patients with cirrhosis without HCC (0.74 [0.4–1.41] pg/mL, p = 0.18) and healthy
controls (0.21 [0.10–0.64] pg/mL). The difference was statistically significant only while comparing HCC patients and controls (p = 0.027). Eight patients with cirrhosis and HCC developed PVT during the follow-up period. These patients showed considerably higher median plasma levels of annexin V-MP (3147 [2690–4272] MP/μL) and endothelial-derived MP (1796 [1098–2454] MP/μL) than patients with cirrhosis and HCC who did not develop PVT (2345 [1422–3877] MP/μL, p = 0.039, and 621 [394–1165] MP/μL, p = 0.012, respectively). They also showed significantly shorter median phospholipid-dependent clotting time (38 [36–55] sec) compared to HCC patients without PVT (68 [52–77] sec, p = 0.001). No difference in MP TF activity was observed in patients neither with nor without PVT (0.92 [0.62–1.11] and 0.80 [0.49–0.92], respectively, p = 0.24). The 95th percentile of all annexin V and endothelial-derived MP was calculated in HCC patients
Fig. 1. Circulating microparticles plasma levels in cirrhosis patients with and without hepatocellular carcinoma (HCC). A): Annexin V-microparticles; B): endothelial-derived MP; C): platelet-derived MP; D): leukocyte-derived MP; E): TF + MP; F) TM + MP. Data are expressed by median and interquartile range. Grey area refers to MP plasma levels found in healthy controls. p calculated between cirrhosis with HCC and cirrhosis without HCC ***b0.001, **b0.005, *b0.05. p calculated between cirrhosis without HCC and controls †††b0.001; ††b0.01; †b0.5.
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who did not develop PVT. Two of the 8 patients with PVT (25%) and four of the 25 without PVT (16%) had MP levels above this cut-off point; this different prevalence was not statistically significant (Chi-square 0.33, p = 0.56) with a RR of 1.50 (95% CI 0.40 to 5.69). Patients with cirrhosis and HCC who developed PVT showed higher median plasma levels of platelet and leukocyte-derived MP, TF + MP and TM + MP than patient without PVT but the differences were not statistically significant. In this study we showed that patients with cirrhosis and HCC had increased annexin V-MP, endothelial, platelet and leukocyte-derived MP, TF + MP, TM + MP, PPL activity and MP TF activity compared to patients with cirrhosis without HCC and healthy controls. Moreover, patients with HCC and cirrhosis who developed PVT showed significantly higher levels of annexin V MP and endothelial-derived MP than patients with cirrhosis and HCC who did not. However circulating levels of MP were associated with a barely higher RR for PVT. Finally, we found that cirrhotic patients without HCC also showed significantly higher median levels of annexin V-MP, endothelial, platelet and leukocyte-derived MP and TM + MP compared to healthy controls. No difference in TF + MP and TF MP activity was found between cirrhosis without HCC and controls. Our results on cancer are in line with previous findings showing that MP levels in patients with Hepatitis C and HCC were higher compared to those with Hepatitis C alone and dynamically decrease by the end of the second week after surgery [10]. In addition, we confirmed the presence of MP with prothrombotic potential in HCC as well, as previously described in other neoplasms [5]. As far as cirrhosis alone is concerned, Rautou et al. [8] showed that MP TF activity was higher in 33 cirrhotic patients than in controls and correlated with disease severity. They also recently showed [9] that hepatocyte TF activates coagulation in a mouse model of chronic liver injury. Moreover, Kornek et al. [11] concluded that MP from immune cells may represent a possible biomarker for the extent and characteristics of hepatic inflammation in patients with chronic liver disease. We confirmed the presence of MP in cirrhotic liver compared to healthy controls, thus asserting a role for endothelial, platelet and leukocyte-MP as mediators in coagulation activation, endothelial injury and pro-inflammatory reactions that are all involved in the hepatic fibrogenesis process. Conversely, we did not detect any difference in MP TF activity and TF + MP in cirrhotic patients, probably because the majority of our patients had stable liver disease (Child Pugh A and B) and only four developed thrombotic complications. It could be possible that in a more severe cirrhotic disease, hepatocyte injury might trigger hepatocyte expression of procoagulant TF and TF + MP production, as showed by Rautou [9]. The strengths of our study lie in the recruitment of a cohort of cirrhotic patients that was prospectively followed for the occurrence of PVT, in the measurement of different MP subtypes involved in endothelial and platelet activation, inflammation and activation of coagulation cascade. In particular we found a global activation of coagulation with the presence of both TF + MP (prothrombotic potential) and TM + MP (anticoagulant potential). Our study suffers from some limitations though. Firstly, the size of the study population is limited, mainly because we only enrolled and prospectively followed cirrhotic patients free of other pathologic conditions potentially associated with an increase in MP levels. This could explain why RR for PVT was not statistically significant. Moreover, the small number of PVT recorded in HCC group (n. 8) did not allow us a subgroup evaluation of any existing association between clinical/molecular characteristics of the neoplasm or chemotherapy treatment and procoagulant MP subtypes. We were still able to observe some considerable differences between the groups that will need to be confirmed in larger studies. Second, old flow cytometric assays may not be sensitive enough to detect all sizes of MP, given that many of these fall below the detection threshold, though recommendations by the ISTH SSC Working Group on Vascular Biology [12] were used both for pre-analytical and for analytical conditions. In addition, we did not perform a multiplex fluorescence analysis to detect the co-expression between
TF and a leukocyte surface marker in order to evaluate the TF activity of the white blood cells compartment and did not analyse the eventual presence of MP derived from hepatocytes. In conclusion, patients with concomitant cirrhosis and HCC showed higher levels of MP and higher MP TF activity than patients with cirrhosis without HCC and healthy controls; patients with HCC and cirrhosis who developed PVT showed significantly higher median plasma levels of annexin V and endothelial-derived MP than patients who did not develop PVT; circulating MP are present in cirrhotic patients but hypercoagulability as assessed by TF + MP and MP TF activity is higher in presence of HCC and cirrhosis than in presence of cirrhosis alone, the discriminating factor being HCC. Conflicts of interest None to declare. Acknowledgements We are indebted to E. F. Nde for his very helpful contribution with the English language. References [1] A. Forner, J.M. Llovet, J. Bruix, Hepatocellular carcinoma, Lancet 379 (2012) 1245–1255. [2] J. Gong, R. Jaiswal, P. Dalla, F. Luk, M. Bebawy, Microparticles in cancer: a review of recent developments and the potential for clinical application, Semin. Cell Dev. Biol. 40 (2015) 35–40. [3] N.S. Barteneva, E. Fasler-Kan, M. Bernimoulin, J.N. Stern, E.D. Ponomarev, L. Duckett, et al., Circulating microparticles: square the circle, BMC Cell Biol. 14 (2013) 23, http://dx.doi.org/10.1186/1471-2121-14-23. [4] S. Nomura, M. Shimizu, Clinical significance of procoagulant microparticles, J. Interv. Cardiol. 3 (2) (2015)http://dx.doi.org/10.1186/s40560-014-0066-z. [5] C. Gardiner, P. Harrison, M. Belting, A. Böing, E. Campello, B.S. Carter, et al., Extracellular vesicles, tissue factor, cancer and thrombosis - discussion themes of the ISEV 2014 educational day, J. Extracell Vesicles 4 (2015) 26901, http://dx.doi.org/10. 3402/jev.v4.26901. [6] E. Campello, L. Spiezia, C.M. Radu, C. Bulato, S. Gavasso, D. Tormene, et al., Circulating microparticles and the risk of thrombosis in inherited deficiencies of antithrombin, protein C and protein S, Thromb. Haemost. 115 (2015) 81–88. [7] E. Campello, L. Spiezia, C.M. Radu, S. Gavasso, B. Woodhams, P. Simioni, Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity, Blood Coagul. Fibrinolysis 25 (2014) 534–537. [8] P.E. Rautou, A.C. Vion, J. Luyendyk, N. Mackman, Circulating microparticles tissue factor activity is increased in patients with cirrhosis, Hepatology 60 (2014) 1793–1795. [9] P.E. Rautou, K. Tatsumi, S. Antoniak, A.P. Owens 3rd, E. Sparkenbaugh, L.A. Holle, et al., Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury, J. Hepatol. 64 (2016) 53–59. [10] S.V. Brodsky, M.E. Facciuto, D. Heydt, J. Chen, H.K. Islam, M. Kajstura, et al., Dynamics of circulating microparticles in liver transplant patients, J. Gastrointestin Liver Dis. 17 (2008) 261–268. [11] M. Kornek, M. Lynch, S.H. Mehta, M. Lai, M. Exley, N.H. Afdhal, et al., Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis, Gastroenterology 143 (2012) 448–458. [12] R. Lacroix, C. Judicone, M. Mooberry, M. Boucekine, N.S. Key, F. Dignat-George, The ISTH SSC Workshop, Standardization of pre-analytical variables in plasma microparticle determination: results of the international society on thrombosis and Haemostasis SSC collaborative workshop, J. Thromb. Haemost. 11 (2013) 1190–1193.
Elena Campello Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Alberto Zanetto Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Luca Spiezia Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy
Correspondence
Claudia M. Radu Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Sabrina Gavasso Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Alberto Ferrarese Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Fabio Farinati Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy
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Marco Senzolo Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Paolo Simioni⁎ Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Corresponding author at: Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Via Ospedale Civile 105, 35100 Padua, Italy. E-mail address: [email protected] 21 February 2016 Available online 20 May 2016
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Thrombosis Research journal homepage: www.elsevier.com/locate/thromres
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Hypercoagulability detected by circulating microparticles in patients with hepatocellular carcinoma and cirrhosis Dear Editors, Portal vein thrombosis (PVT) is a common complication of hepatocellular carcinoma (HCC) and is associated with a poor prognosis [1]. However, studies on the hypercoagulable state associated with this neoplasm and its correlation with the risk of PVT are lacking. Microparticles (MP), a population of extracellular vesicles ranging in size from 0.1 to 1 μm, are released from the surface of cells by the process of outward membrane budding through a loss of calcium-dependent membrane phospholipid asymmetry and cytoskeletal rearrangement [2–4]. MP have been extensively investigated for their ability to induce coagulation and participate in thrombosis because of the presence of tissue factor (TF) and the exposure of negatively charged phospholipids [2–4]. Thus far, numerous studies have analysed levels of circulating MP in patients presenting diseases with a high risk for thromboembolic complications [3,5], but there is not enough data available in Literature regarding the presence of MP in HCC and their potential prothrombotic role. Our aim was to investigate the presence and cellular origins of circulating MP in plasma of patients with cirrhosis with and without HCC and compare them with a healthy control population and to evaluate the possible contribution of MP in PVT occurrence in HCC. All consecutive patients presenting established cirrhosis with or without HCC admitted to the Department of Gastroenterology of University of Padua from January to September 2013 were enrolled into this study. Severity of cirrhosis was estimated according to the ChildPugh classification and MELD score. A control group of 50 consecutive healthy subjects matched for age (± 3 years), sex and ethnic origins was recruited. Exclusion criteria were: ongoing antiplatelet/anticoagulant treatment, inherited thrombophilia, undergoing antiviral treatment, dialysis and extrahepatic neoplasms. This study was approved by the Ethical Committee of Padua Medical Hospital. Written informed consent was obtained from each patient and control. Both patients with and without HCC were followed up for 12 months after the recruitment for the occurrence of PVT. Fasting whole blood samples (9 mL) were drawn at enrolment by clean venipuncture from an antecubital vein and collected into vacuum tubes containing109 mM trisodium citrate. Platelet-free plasma was prepared within 3 h by double centrifugation (2 × 15 min at 2500 g) at room temperature. Aliquots were immediately frozen and then stored at − 80 °C until use. Samples were thawed by incubation for 5 min in a water bath at 37 °C immediately before testing and analysed only after a single freeze-thaw cycle. Assays were performed at least 1 month after storage at −80 °C [6]. Classical coagulation tests were performed (platelet count, activated partial thromboplastin time and prothrombin time, factor II, fibrinogen,
http://dx.doi.org/10.1016/j.thromres.2016.05.021 0049-3848/© 2016 Elsevier Ltd. All rights reserved.
factor VIII, and antithrombin activity, protein C antigen, chromogenic and coagulometric activities, protein S coagulometric activity and total and free antigens) as previously described [6]. MP analysis was performed on a Cytomics FC500 flow cytometer (Beckman Coulter, Miami Florida), as previously described [6]. MP expressing phosphatidylserine were identified by size and annexin V-fluorescein isothiocyanate (FITC) (Bender MedSystems GmbH, Vienna, Austria) labelling (annexinV-MP). The different populations of MP were measured co-labelling with antibodies against cell-type specific antigens and annexin V. Endothelial-derived MP were identified using CD62E-PC5 (phycoerythrin-cyanin5.1), platelet-derived MP using CD62P-PE (phycoerythrin) (both Beckman Coulter, Miami, Florida); leukocyte MP using CD45-PC5 (BioLegend Europe, The Netherlands); tissue factor-bearing (TF + MP) with CD142-PE and thrombomodulin-bearing MP with CD141-PE - both from BD, Biosciences, Milan, Italy. Isotype controls were used. Procoagulant activity of the MP was measured using the STA®Procoag PPL assay (Diagnostica Stago, Asnieres, France) [7]. MP TF activity was measured by an “in-house” endpoint assay using a curve generated with commercially available Innovin™ (Dade Behering) as standard, as previously reported by other groups [8,9]. Normally distributed variables were summarized as mean (± SD) and Student's t-test was performed, whereas non-normally distributed variables were summarized as medians with interquartile ranges (IQR) and Mann-Whitney U test was conducted. For multiple comparisons Kruskall-Wallis test was used. Spearman's correlation analysis was used to detect significant correlations. A “p” value b 0.05 was considered statistically significant. The relative risk (RR) and the 95% confidence intervals (CI) were ascertained from contingency tables (PASW Statistics 17.0.2 for Windows). Sixty-five adult cirrhotic patients [Child: A 21, B 27, C 17], 33 with HCC (M/F 25/8; mean age 64.5 ± 12.5 years) and 32 without HCC (M/ F 24/8; mean age 56 ± 14.7 years) were prospectively enrolled. Median MELD was 11 [8–14] in patients with HCC and 13 [10−20] in patients without HCC, respectively (p = 0.13). Portal vein thrombosis was detected in 12 (18%) cirrhotic patients, 8 with HCC and 4 without HCC. Fifty adult healthy subjects [M/F 25/25, age 54 ± 8 years] acted as controls. Patients with cirrhosis and HCC had a significantly higher platelets count and significantly lower protein C coagulometric activity and antigen thancirrhotic patients without HCC (Table 1). Patients with cirrhosis and HCC showed significantly higher median plasma levels of annexin V-MP (p b 0.001), endothelial-derived MP (p b 0.001), platelet-derived MP (0 b 0.005), leukocyte-derived MP (p b 0.001), TF + MP (p b 0.001) and thrombomodulin (TM) + MP (p b 0.05) than cirrhotic patients without HCC. Moreover, cirrhosis without HCC showed significantly higher levels of annexin V-MP (p b 0.01), endothelial-derived MP (p b 0.001), platelet-derived MP (p = 0.02), leukocyte-derived MP (p b 0.001), and TM + MP (p b 0.001) compared to healthy controls. No significant difference was detected for TF + MP between healthy controls and cirrhotic patients without HCC (p = 0.9) (Fig. 1). Considering the overall study
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Table 1 Basal characteristics of the study population.
Age, years (mean ± SD) Etiology: alcohol-virus/other, n CHILD A/B/C, n MELD (median [IQR]) αFP, μg/L (median [IQR]) Portal vein thrombosis, n (%) Time of PVT occurrence after enrolment, months (median [IQR]) Platelets/μL (mean ± SD) Protein C Chromogenic activity, % Coagulometric activity, % Antigen, % TACE (n of procedures in n patients) Percutaneous RF (n of procedures in n patients) Laparoscopic RF (n of procedures in n patients) Sorafenib treatment (n of patients)
Cirrhosis with HCC (n = 33)
Cirrhosis without HCC (n = 32)
p
64.5 ± 12.5 30/3 13/12/8 11 [8–14] 4.5 [2.3–9.5] 8 (24) 7 [6–8] 127 ± 77
56.0 ± 14.7 20/12 8/15/9 13 [10–20] 4.0 [2.3–9.3] 4 (12.5) 7 [6–9] 97 ± 54
ns ns ns ns ns b0.01 ns 0.046
45.0 ± 31.6 39.8 ± 27.1 45.9 ± 29.8 24 in 22 patients 8 in 6 patients 6 in 5 patients 0
56.4 ± 29.8 53.4 ± 25.2 60.8 ± 29.5
ns 0.029 0.035
HCC, hepatocellular carcinoma; αFP, alphafetoprotein; MELD, model for end stage liver disease; IQR, interquartile range, PVT, portal vein thrombosis; TACE, trans-arterial chemo-embolization; RF, radiofrequency.
population no statistically significant differences in MP plasma levels were detected among patients in different Child-Pugh classes. Median phospholipid-dependent clotting time (PPL assay) was significantly shorter in patients with cirrhosis and HCC (52.4 [46.2–70.0] sec) than in patients with cirrhosis without HCC (74.3 [67.8–79.6] sec, p = 0.001) and healthy controls (74.3 [67.8–80.1] sec, p = 0.007). This shortening of the PPL clotting time reflects an increase procoagulant activity due to phospholipid presence. No difference in PPL clotting time was detected between cirrhosis without HCC and controls (p = 0.81). In the overall study population, the levels of annexin V-MP were inversely correlated with PPL clotting time (r = − 0.66, p = 0.002). Median MP TF activity was higher in patients with concomitant cirrhosis and HCC (1.12 [0.9–1.62] pg/mL) than in patients with cirrhosis without HCC (0.74 [0.4–1.41] pg/mL, p = 0.18) and healthy
controls (0.21 [0.10–0.64] pg/mL). The difference was statistically significant only while comparing HCC patients and controls (p = 0.027). Eight patients with cirrhosis and HCC developed PVT during the follow-up period. These patients showed considerably higher median plasma levels of annexin V-MP (3147 [2690–4272] MP/μL) and endothelial-derived MP (1796 [1098–2454] MP/μL) than patients with cirrhosis and HCC who did not develop PVT (2345 [1422–3877] MP/μL, p = 0.039, and 621 [394–1165] MP/μL, p = 0.012, respectively). They also showed significantly shorter median phospholipid-dependent clotting time (38 [36–55] sec) compared to HCC patients without PVT (68 [52–77] sec, p = 0.001). No difference in MP TF activity was observed in patients neither with nor without PVT (0.92 [0.62–1.11] and 0.80 [0.49–0.92], respectively, p = 0.24). The 95th percentile of all annexin V and endothelial-derived MP was calculated in HCC patients
Fig. 1. Circulating microparticles plasma levels in cirrhosis patients with and without hepatocellular carcinoma (HCC). A): Annexin V-microparticles; B): endothelial-derived MP; C): platelet-derived MP; D): leukocyte-derived MP; E): TF + MP; F) TM + MP. Data are expressed by median and interquartile range. Grey area refers to MP plasma levels found in healthy controls. p calculated between cirrhosis with HCC and cirrhosis without HCC ***b0.001, **b0.005, *b0.05. p calculated between cirrhosis without HCC and controls †††b0.001; ††b0.01; †b0.5.
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who did not develop PVT. Two of the 8 patients with PVT (25%) and four of the 25 without PVT (16%) had MP levels above this cut-off point; this different prevalence was not statistically significant (Chi-square 0.33, p = 0.56) with a RR of 1.50 (95% CI 0.40 to 5.69). Patients with cirrhosis and HCC who developed PVT showed higher median plasma levels of platelet and leukocyte-derived MP, TF + MP and TM + MP than patient without PVT but the differences were not statistically significant. In this study we showed that patients with cirrhosis and HCC had increased annexin V-MP, endothelial, platelet and leukocyte-derived MP, TF + MP, TM + MP, PPL activity and MP TF activity compared to patients with cirrhosis without HCC and healthy controls. Moreover, patients with HCC and cirrhosis who developed PVT showed significantly higher levels of annexin V MP and endothelial-derived MP than patients with cirrhosis and HCC who did not. However circulating levels of MP were associated with a barely higher RR for PVT. Finally, we found that cirrhotic patients without HCC also showed significantly higher median levels of annexin V-MP, endothelial, platelet and leukocyte-derived MP and TM + MP compared to healthy controls. No difference in TF + MP and TF MP activity was found between cirrhosis without HCC and controls. Our results on cancer are in line with previous findings showing that MP levels in patients with Hepatitis C and HCC were higher compared to those with Hepatitis C alone and dynamically decrease by the end of the second week after surgery [10]. In addition, we confirmed the presence of MP with prothrombotic potential in HCC as well, as previously described in other neoplasms [5]. As far as cirrhosis alone is concerned, Rautou et al. [8] showed that MP TF activity was higher in 33 cirrhotic patients than in controls and correlated with disease severity. They also recently showed [9] that hepatocyte TF activates coagulation in a mouse model of chronic liver injury. Moreover, Kornek et al. [11] concluded that MP from immune cells may represent a possible biomarker for the extent and characteristics of hepatic inflammation in patients with chronic liver disease. We confirmed the presence of MP in cirrhotic liver compared to healthy controls, thus asserting a role for endothelial, platelet and leukocyte-MP as mediators in coagulation activation, endothelial injury and pro-inflammatory reactions that are all involved in the hepatic fibrogenesis process. Conversely, we did not detect any difference in MP TF activity and TF + MP in cirrhotic patients, probably because the majority of our patients had stable liver disease (Child Pugh A and B) and only four developed thrombotic complications. It could be possible that in a more severe cirrhotic disease, hepatocyte injury might trigger hepatocyte expression of procoagulant TF and TF + MP production, as showed by Rautou [9]. The strengths of our study lie in the recruitment of a cohort of cirrhotic patients that was prospectively followed for the occurrence of PVT, in the measurement of different MP subtypes involved in endothelial and platelet activation, inflammation and activation of coagulation cascade. In particular we found a global activation of coagulation with the presence of both TF + MP (prothrombotic potential) and TM + MP (anticoagulant potential). Our study suffers from some limitations though. Firstly, the size of the study population is limited, mainly because we only enrolled and prospectively followed cirrhotic patients free of other pathologic conditions potentially associated with an increase in MP levels. This could explain why RR for PVT was not statistically significant. Moreover, the small number of PVT recorded in HCC group (n. 8) did not allow us a subgroup evaluation of any existing association between clinical/molecular characteristics of the neoplasm or chemotherapy treatment and procoagulant MP subtypes. We were still able to observe some considerable differences between the groups that will need to be confirmed in larger studies. Second, old flow cytometric assays may not be sensitive enough to detect all sizes of MP, given that many of these fall below the detection threshold, though recommendations by the ISTH SSC Working Group on Vascular Biology [12] were used both for pre-analytical and for analytical conditions. In addition, we did not perform a multiplex fluorescence analysis to detect the co-expression between
TF and a leukocyte surface marker in order to evaluate the TF activity of the white blood cells compartment and did not analyse the eventual presence of MP derived from hepatocytes. In conclusion, patients with concomitant cirrhosis and HCC showed higher levels of MP and higher MP TF activity than patients with cirrhosis without HCC and healthy controls; patients with HCC and cirrhosis who developed PVT showed significantly higher median plasma levels of annexin V and endothelial-derived MP than patients who did not develop PVT; circulating MP are present in cirrhotic patients but hypercoagulability as assessed by TF + MP and MP TF activity is higher in presence of HCC and cirrhosis than in presence of cirrhosis alone, the discriminating factor being HCC. Conflicts of interest None to declare. Acknowledgements We are indebted to E. F. Nde for his very helpful contribution with the English language. References [1] A. Forner, J.M. Llovet, J. Bruix, Hepatocellular carcinoma, Lancet 379 (2012) 1245–1255. [2] J. Gong, R. Jaiswal, P. Dalla, F. Luk, M. Bebawy, Microparticles in cancer: a review of recent developments and the potential for clinical application, Semin. Cell Dev. Biol. 40 (2015) 35–40. [3] N.S. Barteneva, E. Fasler-Kan, M. Bernimoulin, J.N. Stern, E.D. Ponomarev, L. Duckett, et al., Circulating microparticles: square the circle, BMC Cell Biol. 14 (2013) 23, http://dx.doi.org/10.1186/1471-2121-14-23. [4] S. Nomura, M. Shimizu, Clinical significance of procoagulant microparticles, J. Interv. Cardiol. 3 (2) (2015)http://dx.doi.org/10.1186/s40560-014-0066-z. [5] C. Gardiner, P. Harrison, M. Belting, A. Böing, E. Campello, B.S. Carter, et al., Extracellular vesicles, tissue factor, cancer and thrombosis - discussion themes of the ISEV 2014 educational day, J. Extracell Vesicles 4 (2015) 26901, http://dx.doi.org/10. 3402/jev.v4.26901. [6] E. Campello, L. Spiezia, C.M. Radu, C. Bulato, S. Gavasso, D. Tormene, et al., Circulating microparticles and the risk of thrombosis in inherited deficiencies of antithrombin, protein C and protein S, Thromb. Haemost. 115 (2015) 81–88. [7] E. Campello, L. Spiezia, C.M. Radu, S. Gavasso, B. Woodhams, P. Simioni, Evaluation of a procoagulant phospholipid functional assay as a routine test for measuring circulating microparticle activity, Blood Coagul. Fibrinolysis 25 (2014) 534–537. [8] P.E. Rautou, A.C. Vion, J. Luyendyk, N. Mackman, Circulating microparticles tissue factor activity is increased in patients with cirrhosis, Hepatology 60 (2014) 1793–1795. [9] P.E. Rautou, K. Tatsumi, S. Antoniak, A.P. Owens 3rd, E. Sparkenbaugh, L.A. Holle, et al., Hepatocyte tissue factor contributes to the hypercoagulable state in a mouse model of chronic liver injury, J. Hepatol. 64 (2016) 53–59. [10] S.V. Brodsky, M.E. Facciuto, D. Heydt, J. Chen, H.K. Islam, M. Kajstura, et al., Dynamics of circulating microparticles in liver transplant patients, J. Gastrointestin Liver Dis. 17 (2008) 261–268. [11] M. Kornek, M. Lynch, S.H. Mehta, M. Lai, M. Exley, N.H. Afdhal, et al., Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis, Gastroenterology 143 (2012) 448–458. [12] R. Lacroix, C. Judicone, M. Mooberry, M. Boucekine, N.S. Key, F. Dignat-George, The ISTH SSC Workshop, Standardization of pre-analytical variables in plasma microparticle determination: results of the international society on thrombosis and Haemostasis SSC collaborative workshop, J. Thromb. Haemost. 11 (2013) 1190–1193.
Elena Campello Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Alberto Zanetto Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Luca Spiezia Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy
Correspondence
Claudia M. Radu Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Sabrina Gavasso Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Alberto Ferrarese Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Fabio Farinati Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy
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Marco Senzolo Multivisceral Transplant Unit, Department of Surgical and Gastroenterological Sciences, University of Padua Medical School, Padua, Italy Paolo Simioni⁎ Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Padua, Italy Corresponding author at: Thrombotic and Hemorrhagic Diseases Unit, Department of Medicine, University of Padua Medical School, Via Ospedale Civile 105, 35100 Padua, Italy. E-mail address: [email protected] 21 February 2016 Available online 20 May 2016