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PO-54 Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay and an automated procoagulant assay
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5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166–S191
thrombogram, CAT) and the PCA (by the P-PPL/1 assay), of plasma MP in 15 ET (5M/10F) and 15 matched control (C) (5M/10F) subjects. Materials and Methods: Platelet Free Plasma (PFP) was obtained by two serial centrifugations (4,000 rpm for 15 min, 11,000 rpm for 10 min) and MP-free plasma (MP-FP) by re-centrifuging PFP at 14,000 rpm for 30 min. MP were isolated from the pellet. For CAT assay, 80 ml of P-FP or MP-FP and 20 ml of buffer were mixed, and TG started adding CaCl2. The results were expressed as LagTime, Peak, area under the curve (ETP), and Time-to-Peak (ttPeak). For P-PPL/1 assay, 100 ml of P-FP or MP-FP were mixed with 50 ml of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds. Results: PFP from ET patients produced significantly (p < 0.05) higher TG (LagTime: 14.8±3.5 min) and PCA (76.7±8.1 sec) then controls (LagTime: 20.5±7.9 min; PCA: 89.2±7.6 sec). This increase was due to the presence of MP, as no TG and PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples, which was identical to the original values of PFP for both assays. There was a significant correlation between PCA by the PPPL/1 assay and the different parameters of TG assay [lagtime (R2 = 0.749), Peak (R2 = −0.809), ETP (R2 = −0.745) and TTPeak (R2 = −0.773)]. Conclusions: The hypercoagulable state of ET patients is characterized by an increased MP-associated PCA. Prospective studies are needed to evaluate whether MP-associated TG and PCA can predict for thrombosis in these patients. PO-53 Platelet derived microparticles induce factor XII mediated thrombin generation H.M. Spronk1 *, E. Kilinc1 , R. van Oerle1 , K. Hamulyak1 , T. Renne2 , H. ten Cate1 . 1 Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, Cardiovascular Research Institue Maastricht, Maastricht University, The Netherlands, 2 Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Introduction: Microparticles are small (<1 mm) cell derived vesicles expressing antigens from the ancestor cell at their outer surface. Due to the presence of a procoagulant phospholipid bilayer as well as tissue factor, the main physiological activator of coagulation, microparticles have been linked to the hypercoagulable state in cancer patients. Given the recently described activation of factor XII by platelet derived polyphosphates (polyP), we hypothesized that platelet derived microparicles induce thrombin generation through activation of factor XII. Methods: Isolated human platelets were stimulated by ADP and ionophore or ionophore alone, whereas monocytes were activated by ionophore with and without lipopolysacharide (LPS) activation. Generated microparticles were isolated and characterised for expression of antigens through FACS analysis, tissue factor activity, and quantified using a newly developed ELISA-based method. Results: Platelet derived microparticles expressed mainly antigens also present on platelets and had low tissue factor activity of <1 pM. Microparticles from LPS stimulated monocytes, however, displayed a higher tissue factor activity (>500 pM) as expected. Using a GP1b-specific antibody and detection through an antibody against GPIIb (CD41) alowed for quantification of platelet derived microparticles. Addition of plateletderived microparticles to human normal pooled plasma induced thrombin generation characterised by a lag time of >10 min and a peak height of 100 nM thrombin. Inhibition of the tissue factor pathway, using active site inhibited seven (ASIS), had no effect on thrombin generation. In contrast, platelet derived microparticles failed to induce thrombin generation in factor XII-deficient plasma. Monocyte derived microparticle induced thrombin generation, however, was comparable between normal and fXIIdeficient plasma and almost completely abolished in the presence of ASIS. Conclusions: Monocyte derived microparticles mainly induced thrombin generation through tissue factor, whereas microparticles from platelets activated factor XII mediated coagulation.
PO-54 Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay and an automated procoagulant assay F. Debaugnies1 *, M.A. Azerad2,3 , D. Noubouossie´ 1 , L. Rozen1 , H.C. Hemker4 , A. Efira2 , A. Demulder1 . 1 Laboratory of Hematology and Haemostasis, CHU Brugmann, ULB, 2 Hemato-Oncology Clinic CHU Brugmann, ULB, 3 Jules Bordet Institute, ULB, Brussels, Belgium, 4 Synapse BV, Cardiovascular Research Institute CARIM, Maastricht, The Netherlands Introduction: Circulating microparticles (MP) are reported to play a role in cancer hypercoagulability. The procoagulant properties of MP derive from the amount of tissue factor and/or phosphatidylserine that they carry. Aim: To assess the procoagulant activity of MP in the plasma of newly diagnosed cancer patients with a simple assay, easy to implement in the laboratory. Patients and Methods: Patients (n = 31): newly diagnosed cancer patients without anticoagulant or chemotherapy compared to matched controls. We used a thrombin generation (TG) assay, i.e. by the CAT® method, in four conditions: 1: addition of 1 pM tissue factor (TF) and 4 mM procoagulant phospholipids (PPL) (positive control), 2: without any trigger, 3: addition of TF only, 4: addition of PPL only. In parallel, we tested an automated activated factor X-based clotting assay depending on PPL activity (STA®Procoag-PPL). Results: When we added only PPL (see table), so that TG is dependent upon endogenous TF only, tlag was significantly shorter in cancer patients. When we added only TF, i.e. made the results dependent upon PPL, ETP, peak, and velocity index were significantly higher and ttp was significantly shorter. This suggests that the procoagulant activity in cancer patients is provided by both PL-bearing MP and TF-bearing MP. Concerning the clotting assay, we observed that patients who had the shortest clotting times (mainly gastro-intestinal tract and breast cancers), corresponded to patients with the highest peak and velocity index parameters of the TG using TF alone. Conclusions: CAT® method is a simple method that demonstrates PPL and TF activity in the plasma of newly diagnosed cancer patients, and brings potentially useful information that other methods cannot provide. Further experiments are needed to enhance the specificity of the test (contribution of intrinsic pathway, excess of FVIII . . . ). Further patient samples are needed to establish a comparison between CAT® and STA®-Procoag-PPL. TG assay
Tlag (%) ETP (%) Peak (%) Ttp (%) Velocity Index (%)
Baseline controla Cancer Matched patients controls
TF added only Cancer patients
Matched controls
PPL added only Cancer patients
Matched controls
296 (231–382) 86 (80–94)** 54 (41–72)** 232 (195–282) 39 (28–65)**
136 (128–156) 98 (90–108)* 57 (46–78)*** 146 (127–159)*** 37 (27–61)***
144 (136–157) 90 (79–99) 41 (30–49) 175 (155–181) 21 (14–25)
240 (190–297)** 86 (83–93) 94 (86–105) 183 (152–216) 107 (86–122)
285 (260–300) 91 (83–97) 100 (92–105) 195 (180–210) 117 (95–131)
343 (311–386) 76 (62–86) 41 (30–48) 250 (226–279) 28 (20–38)
a Without addition of TF or PPL. The parameters of the thrombogram, ETP (endogenous thrombin potential), Peak (maximum thrombin concentration), Tlag (lag time) and Ttp (time to peak) as well as the velocity index [= peak/(Ttp − Tlag)] were expressed as a % of the positive control, reported as median (25–75 percentile). Comparison between cancer patients and matched controls using Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001.
Clotting time (s), Procoag PPL Peak (nM), TG Velocity index (nM/min), TG
Cancer patients with clotting time <41 s (n = 10)
Other cancer patients (n = 19)
Controla (n = 31)
34 (31–37)* 297 (267–330)* 77 (70–101)*
48 (45–55) 173 (147–229) 44 (28–56)
46 (41–50) 142 (112–188) 27 (19–38)
Bonferroni’s multiple comparison test: *p < 0.01 (patients with clotting time <41 s vs other cancer patients and vs controls).
PO-55 Treatment of the monocytic leukemia cell line THP-1 with anthracyclines increases cellular tissue factor activity and release of tissue factor positive microparticles J. Boles *, D. Barcel, A.P. Owens III, S. Glover, R. Kasthuri, N. Key, N. Mackman. Division of Hematology/Oncology Department of Medicine University of North Carolina, Chapel Hill, NC, USA Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. While malignancy alone conveys an increased risk for thrombosis, treatment with cytotoxic chemotherapeutics further elevates this risk. However, the pathogenic mechanisms underlying this process remain
5th ICTHIC Abstracts: Poster Sessions / Thrombosis Research 125 (2010) S166–S191
thrombogram, CAT) and the PCA (by the P-PPL/1 assay), of plasma MP in 15 ET (5M/10F) and 15 matched control (C) (5M/10F) subjects. Materials and Methods: Platelet Free Plasma (PFP) was obtained by two serial centrifugations (4,000 rpm for 15 min, 11,000 rpm for 10 min) and MP-free plasma (MP-FP) by re-centrifuging PFP at 14,000 rpm for 30 min. MP were isolated from the pellet. For CAT assay, 80 ml of P-FP or MP-FP and 20 ml of buffer were mixed, and TG started adding CaCl2. The results were expressed as LagTime, Peak, area under the curve (ETP), and Time-to-Peak (ttPeak). For P-PPL/1 assay, 100 ml of P-FP or MP-FP were mixed with 50 ml of phospholipid-depleted plasma. Clotting was started by adding FXa and CaCl2 and results expressed in seconds. Results: PFP from ET patients produced significantly (p < 0.05) higher TG (LagTime: 14.8±3.5 min) and PCA (76.7±8.1 sec) then controls (LagTime: 20.5±7.9 min; PCA: 89.2±7.6 sec). This increase was due to the presence of MP, as no TG and PCA was observed in MP-FP from both patients and controls. The addition of isolated MP to autologous MP-FP restored the TG and PCA of the samples, which was identical to the original values of PFP for both assays. There was a significant correlation between PCA by the PPPL/1 assay and the different parameters of TG assay [lagtime (R2 = 0.749), Peak (R2 = −0.809), ETP (R2 = −0.745) and TTPeak (R2 = −0.773)]. Conclusions: The hypercoagulable state of ET patients is characterized by an increased MP-associated PCA. Prospective studies are needed to evaluate whether MP-associated TG and PCA can predict for thrombosis in these patients. PO-53 Platelet derived microparticles induce factor XII mediated thrombin generation H.M. Spronk1 *, E. Kilinc1 , R. van Oerle1 , K. Hamulyak1 , T. Renne2 , H. ten Cate1 . 1 Laboratory for Clinical Thrombosis and Haemostasis, Department of Internal Medicine, Cardiovascular Research Institue Maastricht, Maastricht University, The Netherlands, 2 Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden Introduction: Microparticles are small (<1 mm) cell derived vesicles expressing antigens from the ancestor cell at their outer surface. Due to the presence of a procoagulant phospholipid bilayer as well as tissue factor, the main physiological activator of coagulation, microparticles have been linked to the hypercoagulable state in cancer patients. Given the recently described activation of factor XII by platelet derived polyphosphates (polyP), we hypothesized that platelet derived microparicles induce thrombin generation through activation of factor XII. Methods: Isolated human platelets were stimulated by ADP and ionophore or ionophore alone, whereas monocytes were activated by ionophore with and without lipopolysacharide (LPS) activation. Generated microparticles were isolated and characterised for expression of antigens through FACS analysis, tissue factor activity, and quantified using a newly developed ELISA-based method. Results: Platelet derived microparticles expressed mainly antigens also present on platelets and had low tissue factor activity of <1 pM. Microparticles from LPS stimulated monocytes, however, displayed a higher tissue factor activity (>500 pM) as expected. Using a GP1b-specific antibody and detection through an antibody against GPIIb (CD41) alowed for quantification of platelet derived microparticles. Addition of plateletderived microparticles to human normal pooled plasma induced thrombin generation characterised by a lag time of >10 min and a peak height of 100 nM thrombin. Inhibition of the tissue factor pathway, using active site inhibited seven (ASIS), had no effect on thrombin generation. In contrast, platelet derived microparticles failed to induce thrombin generation in factor XII-deficient plasma. Monocyte derived microparticle induced thrombin generation, however, was comparable between normal and fXIIdeficient plasma and almost completely abolished in the presence of ASIS. Conclusions: Monocyte derived microparticles mainly induced thrombin generation through tissue factor, whereas microparticles from platelets activated factor XII mediated coagulation.
PO-54 Evaluation of the procoagulant activity in the plasma of cancer patients using a thrombin generation assay and an automated procoagulant assay F. Debaugnies1 *, M.A. Azerad2,3 , D. Noubouossie´ 1 , L. Rozen1 , H.C. Hemker4 , A. Efira2 , A. Demulder1 . 1 Laboratory of Hematology and Haemostasis, CHU Brugmann, ULB, 2 Hemato-Oncology Clinic CHU Brugmann, ULB, 3 Jules Bordet Institute, ULB, Brussels, Belgium, 4 Synapse BV, Cardiovascular Research Institute CARIM, Maastricht, The Netherlands Introduction: Circulating microparticles (MP) are reported to play a role in cancer hypercoagulability. The procoagulant properties of MP derive from the amount of tissue factor and/or phosphatidylserine that they carry. Aim: To assess the procoagulant activity of MP in the plasma of newly diagnosed cancer patients with a simple assay, easy to implement in the laboratory. Patients and Methods: Patients (n = 31): newly diagnosed cancer patients without anticoagulant or chemotherapy compared to matched controls. We used a thrombin generation (TG) assay, i.e. by the CAT® method, in four conditions: 1: addition of 1 pM tissue factor (TF) and 4 mM procoagulant phospholipids (PPL) (positive control), 2: without any trigger, 3: addition of TF only, 4: addition of PPL only. In parallel, we tested an automated activated factor X-based clotting assay depending on PPL activity (STA®Procoag-PPL). Results: When we added only PPL (see table), so that TG is dependent upon endogenous TF only, tlag was significantly shorter in cancer patients. When we added only TF, i.e. made the results dependent upon PPL, ETP, peak, and velocity index were significantly higher and ttp was significantly shorter. This suggests that the procoagulant activity in cancer patients is provided by both PL-bearing MP and TF-bearing MP. Concerning the clotting assay, we observed that patients who had the shortest clotting times (mainly gastro-intestinal tract and breast cancers), corresponded to patients with the highest peak and velocity index parameters of the TG using TF alone. Conclusions: CAT® method is a simple method that demonstrates PPL and TF activity in the plasma of newly diagnosed cancer patients, and brings potentially useful information that other methods cannot provide. Further experiments are needed to enhance the specificity of the test (contribution of intrinsic pathway, excess of FVIII . . . ). Further patient samples are needed to establish a comparison between CAT® and STA®-Procoag-PPL. TG assay
Tlag (%) ETP (%) Peak (%) Ttp (%) Velocity Index (%)
Baseline controla Cancer Matched patients controls
TF added only Cancer patients
Matched controls
PPL added only Cancer patients
Matched controls
296 (231–382) 86 (80–94)** 54 (41–72)** 232 (195–282) 39 (28–65)**
136 (128–156) 98 (90–108)* 57 (46–78)*** 146 (127–159)*** 37 (27–61)***
144 (136–157) 90 (79–99) 41 (30–49) 175 (155–181) 21 (14–25)
240 (190–297)** 86 (83–93) 94 (86–105) 183 (152–216) 107 (86–122)
285 (260–300) 91 (83–97) 100 (92–105) 195 (180–210) 117 (95–131)
343 (311–386) 76 (62–86) 41 (30–48) 250 (226–279) 28 (20–38)
a Without addition of TF or PPL. The parameters of the thrombogram, ETP (endogenous thrombin potential), Peak (maximum thrombin concentration), Tlag (lag time) and Ttp (time to peak) as well as the velocity index [= peak/(Ttp − Tlag)] were expressed as a % of the positive control, reported as median (25–75 percentile). Comparison between cancer patients and matched controls using Mann–Whitney test. *p < 0.05, **p < 0.01, ***p < 0.001.
Clotting time (s), Procoag PPL Peak (nM), TG Velocity index (nM/min), TG
Cancer patients with clotting time <41 s (n = 10)
Other cancer patients (n = 19)
Controla (n = 31)
34 (31–37)* 297 (267–330)* 77 (70–101)*
48 (45–55) 173 (147–229) 44 (28–56)
46 (41–50) 142 (112–188) 27 (19–38)
Bonferroni’s multiple comparison test: *p < 0.01 (patients with clotting time <41 s vs other cancer patients and vs controls).
PO-55 Treatment of the monocytic leukemia cell line THP-1 with anthracyclines increases cellular tissue factor activity and release of tissue factor positive microparticles J. Boles *, D. Barcel, A.P. Owens III, S. Glover, R. Kasthuri, N. Key, N. Mackman. Division of Hematology/Oncology Department of Medicine University of North Carolina, Chapel Hill, NC, USA Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. While malignancy alone conveys an increased risk for thrombosis, treatment with cytotoxic chemotherapeutics further elevates this risk. However, the pathogenic mechanisms underlying this process remain